Compositions and methods for the prevention and control of insulin-induced hypoglycemia

ABSTRACT

Pharmaceutical compositions comprising both insulin and glucagon can be administered to control and treat diabetes while reducing or eliminating the risk of insulin-induced hypoglycemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. non-provisionalpatent application Ser. No. ______, entitled “Compositions and Methodsfor the Prevention and Control of Insulin-Induced Hypoglycemia,” filedJun. 23, 2005, attorney docket number 022354-000310US, which is the U.S.National Phase of PCT patent application PCT/US2003/041103 (filed 23Dec., 2003) and which claims priority to U.S. provisional patentapplication Nos. 60/436,735 (filed 27 Dec. 2002), 60/454,972 (filed 14Mar. 2003) and 60/470,346 (filed 13 May 2003). The present applicationfurther claims the benefit of U.S. provisional patent application60/584,449 (filed Jun. 29, 2004). The entirety of each of theapplications is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the fields of biology, pharmacology,and medicine. In particular, the invention relates to compositions andmethods of using compositions for the control of blood glucose levels.

BACKGROUND OF THE INVENTION

Insulin is produced by the beta cells and glucagon by the alpha cells ofthe Pancreatic Islets of Langerhans. One of insulin's major effects isto lower blood glucose by suppressing hepatic glucose output andstimulating peripheral glucose uptake. Endogenous insulin levels may below or undetectable in some patients with diabetes mellitus. Exogenousinsulin is usually administered to reduce hyperglycemia in situationswhere circulating insulin levels are either low or ineffective. Glucagongenerally has effects opposite to those of insulin, including,primarily, increasing hepatic glucose output and thereby increasingblood glucose levels. Glucagon levels tend to increase when bloodglucose levels fall to abnormally low levels, particularly in patientswho utilize exogenous insulin.

Current goals for diabetes management include near normal blood glucoselevels to delay or prevent microvascular complications; achievement ofthis goal usually requires intensive insulin therapy. In striving toachieve this goal, physicians have encountered a substantial increase inthe frequency and severity of hypoglycemia in their diabetic patients.

Hypoglycemia, characterized by low blood glucose levels, results inautonomic and adrenergic, as well as neuroglycopenic, symptoms; thesesymptoms typically are encountered as a result of inadvertent excessiveinsulin administration. Currently, hypoglycemia is defined as a bloodglucose of <70 mg/dl, e.g. greater than 50 or 60 mg/dl. Frequentrecurrent bouts of hypoglycemia can be associated with hypoglycemicunawareness which can further contribute to development of hypoglycemiawhich is sometimes severe. Thus, efforts to achieve normal glucoselevels with insulin can result in the development of hypoglycemia ofvarying frequency and severity in patients. Hypoglycemia and the lack ofawareness of its presence are serious complications of insulin therapythat occur with greater frequency and severity when impairedcounter-regulatory (anti-insulin) responses are present in diabeticpatients. One of the major counter-regulatory hormones that normallyresponds to hypoglycemia is glucagon. Not infrequently, the glucagonresponse to acute hypoglycemia is impaired or lost in patients withadvanced Type 1 and Type 2 diabetes.

SUMMARY OF THE INVENTION

There remains a need for new methods for treating diabetes and newpreparations of insulin and glucagon that reduce the risk ofhypoglycemia induced by insulin therapy. The present invention meetsthis and other needs.

One aspect of the invention provides pharmaceutical compositionscomprising both insulin and glucagon in amounts that can be administeredto a diabetic patient not only to achieve therapeutically effectivecontrol of diabetes but also to prevent hypoglycemia. The formulationscan include, for example, formulations suitable for injection, includingby subcutaneous (s.c.) administration, formulations suitable foradministration orally, formulations suitable for transdermaladministration, formulations suitable for ocular administration, andformulations suitable for inhalation. In one embodiment, thecompositions comprises between about 0.1 to 5 percent glucagon toinsulin by weight for I.V. administration, or a dose equivalent amountfor other methods of administration. In another embodiment, thecompositions comprise about 0.5 to 2 percent glucagon to insulin byweight when the composition is for I.V. administration, or a doseequivalent amount for other methods of administration. For example, insome embodiments, the glucagon is administered subcutaneously (“s.c.”)and 0.1% to 20% glucagon to insulin by weight is administered. In someembodiments, the composition is configured for s.c. administration andcomprises 0.1-20 ng/kg/min. of glucagon to 2-20 units of insulin. Insome embodiments, the composition is configured for s.c. administrationand comprises sufficient glucagon for the administration of 5 to about20 ng/kg/min. (e.g. more than 5 to 20 ng of glucagon for each kg of aperson for each minute of effectiveness) of glucagon. In one embodiment,5 to about 20 ng/kg/min. of glucagon is administered to 1-20 units ofinsulin. Administered ratios can be, for example, administered once anhour. In a preferred embodiment, the composition is suitable foradministration of more 5 to about 20 ng/kg/min of glucagon for each 1-2units of insulin administered. As will be appreciated, in someembodiments, the glucagon and insulin can be kept in separate containersand are not administered at the same time, but the appropriate ratiosbetween the two are maintained. In one embodiment, the separatecontainers are contained in a single device suitable for administrationof the glucagon and insulin, for example for administrationsubcutaneously; in another, two devices are used, one for each agent.

In another aspect, methods to treat diabetes in a human or other mammalwithout inducing or with a substantially reduced risk of inducinghypoglycemia are provided. In one embodiment, the composition comprisinginsulin and glucagon is administered to a patient before the symptoms ofmild, moderate or severe hypoglycemia are present. In some embodiments,the methods of the invention are practiced to prevent nocturnalhypoglycemia in a Type I diabetic patient being treated with insulintherapy, including intensive insulin therapy. The methods compriseco-administration of insulin and glucagon, wherein said insulin isadministered in amounts therapeutically effective for the control ofdiabetes, and said glucagon is administered in amounts therapeuticallyeffective for the prevention of hypoglycemia, and wherein both insulinand glucagon are preferably administered simultaneously with one anotheror contemporaneously with one another, i.e., within about four hours ofeach other (as when regular, LISPRO, and ASPART insulins are used) orwithin about six to twelve hours of each other (as when longer actinginsulins are used), and in any event prior to the onset of clinicallyobservable hypoglycemia. In one embodiment, glucagon is administeredbefore the insulin is administered. In another embodiment, insulin isadministered before glucagon is administered. In one embodiment, themethod involves maintaining the level of blood sugar above 70 mg/dL andbelow 180 mg/dL by the co-administration of insulin and glucagon to adiabetic patient. In another embodiment, the method involvesadministering glucagon s.c. in an amount between about 6 and 18 ng/kgper minute of glucagon. In one embodiment, 1-20 or 2-20 units of insulinare administered to a diabetic patient receiving glucagon in an amountbetween 6 and 18 ng/kg/min s.c. In another embodiment, the methodinvolves administering between about 8 and 12 ng/kg per minute ofglucagon s.c. In one embodiment, 0.1 to 2 or 2-20 units of insulin areadministered to a diabetic patient receiving glucagon in an amountbetween 8 and 12 ng/kg/min. s.c. In another embodiment, the glucagon isadministered by a means other than intravenously or subcutaneously, anda dose equivalent to the s.c. dosing provided above is administered.

In another aspect, methods to maintain blood glucose levels in a rangethat is neither hyperglycemic nor hypoglycemic are provided. Thesemethods comprise the co-administration of insulin and glucagon.

In another aspect, glucagon formulations and modified glucagon suitablefor co-administration with insulin in accordance with the presentmethods are provided.

In another aspect, kits are provided for preventing hypoglycemia. In oneembodiment, the kits preferably include insulin, glucagon, andinstructions for simultaneously administering the appropriatecombination thereof.

In another aspect, the kits include insulin, a long acting form ofglucagon, and instructions for use.

In some aspects, methods for restoring or preventing loss ofhypoglycemic awareness or sensitivity is provided. The methods compriseadministering an amount of glucagon to a patient over a period of timethat is sufficient to prevent or restore hypoglycemic awareness to thepatient. In one embodiment, the patient is administered insulinconcurrently with the administration of glucagon.

In one aspect, a pharmaceutical formulation is provided that comprisesinsulin in an amount effective for the control of diabetes and glucagonin an amount effective for the prevention of hypoglycemia in a human orother mammal. The pharmaceutical formulation is configured to beadministered subcutaneously and the ratio of insulin to glucagon istypically about 1 unit of insulin to between more than 40 milliunits to200 milliunits of glucagon. In some embodiments, the amount of glucagonis between about 50 and 100 milliunits. In some embodiments, theglucagon is a longer-acting form of glucagon. In some embodiments, thelonger-acting form of glucagon contains iodine. In some embodiments, thelonger-acting form of glucagon contains zinc. In some embodiments, thelonger-acting form of glucagon further comprises protamine.

In another aspect, methods of treating diabetes in a human or othermammal without inducing hypoglycemia are provided. The methods compriseadministering insulin in an amount therapeutically effective for thecontrol of diabetes. The insulin can be in an amount between 0.5 and 20Units of insulin. The methods further comprise administering glucagon ina time and an amount therapeutically effective for the prevention ofhypoglycemia. The glucagon can be administered subcutaneously and in anamount between more than 5 and less than or equal to 100 ng per kg ofpatient per minute of desired glucagon effectiveness. In someembodiments, the amount of glucagon administered is between 6 and 18 ngper kg of patient per minute of desired glucagon effectiveness. In someembodiments, the glucagon is a glucagon with a prolonged duration ofaction. In some embodiments, the glucagon is contained in a liposomalformulation. In some embodiments, the glucagon is contained in amicrosphere. In some embodiments, one administers a formulationcomprising both insulin and glucagon. In some embodiments, the insulinand glucagon are contained in a pump that controls administration of adrug to a patient. In some embodiments, the glucagon is administeredsimultaneously with insulin. In some embodiments, the ratio of glucagonto insulin is about more than 40 to 200 milliunits of glucagon to 1 unitof insulin. In some embodiments, 2 units of insulin are administered. Insome embodiments, 10 units of insulin are administered and between 30and 90 ng per kg per minute of glucagon are administered subcutaneously.

In another aspect, kits for the administration of glucagon and insulinin amounts to prevent hypoglycemia is provided. The kits compriseglucagon and insulin. The glucagon and insulin are in a ratio of 1-20units of insulin to 32-480 milliunits of glucagon. The kits furthercomprise a means for administering glucagon subcutaneously andinstructions for the administration of insulin and glucagon so that theglucagon prevents a hypoglycemic event. In some embodiments, theconcentration of glucagon when completely dissolved in a glycerinesolution is more than 500 micrograms per milliliter but less than 2000micrograms per milliliter. In some embodiments, the glucagon and insulinare in a ratio of 1-3 units of insulin to 32-96 milliunits of glucagon.In some embodiments, the means for administering the glucagonsubcutaneously is a pump and said pump is configured to deliver betweenabout 6 to 20 ng/kg/minute of glucagon.

In another aspect, the use of glucagon in combination with insulin inthe preparation of a medicament for treatment of diabetes is provided.Glucagon is used in an amount sufficient to prevent an onset ofhypoglycemia, wherein a ratio of glucagon to insulin is between morethan 40 micrograms and less than 500 micrograms of glucagon to 1-20units of insulin. In some embodiments, the amount is sufficient toprevent an onset of hypoglycemia unawareness. In some embodiments, theamount of insulin is between 1 and 20 units and the amount of glucagonis between 41 and 200 milliunits. In some embodiments, a ratio ofinsulin to glucagon is about between 1 and 3 units of insulin to betweenmore than 40 and less than or equal to about 96 milliunits glucagon. Insome embodiments, the glucagon further comprises protamine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph illustrating idealized pharmacokinetics for a mixtureof regular and intermediate acting (Lente or NPH) insulin.

FIG. 2 is a graph illustrating the insulin profile of a hypotheticalpatient, as described in Example 1, Part A(i), showing a very simple,flat line graph (basal level set by the GLARGINE (LANTUS)) punctuated bypeaks corresponding to prandial LISPRO (HUMALOG) insulin injections.

FIG. 3 is a schematic of a drug delivery pump configured for practice ofan embodiment described in Example 2.

FIG. 4 is a schematic of a drug delivery pump configured for practice ofan embodiment described in Example 2.

FIG. 5 is a schematic of a drug delivery pump configured for practice ofan embodiment described in Example 2.

FIG. 6 illustrates the effect of molecular weight and lipophilicity onthe rate of transdermal transport in case of permeation (upper and lowergray curve for the more or less lipophilic substances, respectively) orof the TRANSFEROME® mediated penetration (black line and bullets).Dotted black bullets represent the commercial drugs in transdermalpatches.

FIG. 7 is a graph illustrating the insulin and glucagon profiles of ahypothetical patient, as described in Example 3, showing for both drugsa very simple, flat line graph (basal insulin and glucagon infusions)punctuated by peaks (corresponding to prandial insulin and glucagoninfusions) corresponding to when glucagon and insulin are administeredin an admixed formulation.

FIG. 8 is a graph illustrating the effect of continuous glucagoninfusion on mean glucagon levels, as described in Example 7.

FIG. 9 is a graph illustrating the effect of continuous glucagoninfusion on mean glucose levels, as described in Example 7.

FIG. 10 is a graph comparing the effect of continuous glucagon infusionat 12 ng/kg/min. on mean glucose and glucagon levels, as described inExample 7.

FIG. 11 is a graph comparing the effect of continuous glucagon infusionat 16 ng/kg/min. on mean glucose and glucagon levels, as described inExample 7.

FIG. 12 is a graph comparing the effect of various doses of glucagon (0,8, and 16 ng/kg/min. of glucagon) on increasing insulin levels (fromabout 1 to about 2.7 units). The graph demonstrates that low doses ofglucagon are capable of preventing insulin induced hypoglycemia, asdescribed in Example 8.

FIG. 13 is a graph displaying the effect of a low dose of glucagon onblood glucose levels and how it can prevent hypoglycemia, as describedin Example 8.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions are provided that can prevent, or significantlyreduce the frequency and severity of, hypoglycemia in insulin-treateddiabetic patients (both Type 1 and 2). In one aspect, the methods andcompositions are employed to treat diabetes while regulating glucoselevels above the levels of hypoglycemia. The methods and compositionscan be used to replenish or restore the abnormally low glucagonresponses often coincident with insulin administration, therebypreventing hypoglycemia.

One issue that complicates the prevention of hypoglycemia is thatrepeated hypoglycemic events can lead to a loss of hypoglycemicawareness; thus, even if initially detected by a patient, the patient'sability to identify hypoglycemic symptoms can be compromised or lostover time. Thus, compositions and methods that can prevent or reversethe loss of hypoglycemic awareness are desirable. One method by whichthis can be achieved is to administer glucagon, or another agent thatelevates levels of blood glucose as described herein, in a relativelylow dose over the time period in which insulin is to act to prevent theonset of mild hypoglycemia. This can also be used to reverse or preventa loss of hypoglycemic awareness.

In one embodiment, the invention provides pharmaceutical formulations oftwo hormones, insulin and glucagon, that are combined in molar ratiosthat optimize glycemic management and attenuate the incidence of orprevent hypoglycemia. In another embodiment, methods and compositionsfor the simultaneous but separate administration of insulin and glucagonto achieve this benefit are provided. While the simultaneousadministration of two hormones with activities viewed as counteractingwould traditionally have appeared to have no beneficial effect, some ofthe present embodiments arise in part from the realization that suchadministration achieves the beneficial effect of preventing hypoglycemiaby virtue of the buffering or blunting effects of glucagon withoutdiminishing the beneficial effects of glucose regulation provided byinsulin. In some embodiments, a low amount of glucagon is continuouslyadministered to a patient that is, has, or is going to receive insulin.Thus, the use of a hyperglycemic agent, such as glucagon, to prevent theonset of hypoglycemia and its associated symptoms due to insulinadministration, is contemplated. In some embodiments, a hyperglycemicagent, such as glucagon, is used to prevent the onset of iatrogenichypoglycemia.

Thus, some of the embodiments provide a method for controlling diabeteswith a reduced risk of hypoglycemia by simultaneous administration ofinsulin and glucagon to a diabetic patient. In one embodiment, a methodof preventing hypoglycemia in a diabetic patient who is being treatedwith insulin and who is not suffering hypoglycemic symptoms is provided,comprising administering glucagon to the patient in an amounttherapeutically effective for the prevention of hypoglycemia. In oneembodiment, the glucagon is administered simultaneously with theinsulin. In another embodiment, the glucagon is administered 10 minutesto hours before additional insulin is administered, and more preferably,30 minutes to 60 minutes before additional insulin is administered. Inother embodiments, the glucagon is administered within about one minuteto about four hours after said patient has last been administeredinsulin. In one embodiment, the prevention of hypoglycemia comprisespreventing the symptoms associated with hypoglycemia from becomingevident in a subject. In another embodiment, the prevention ofhypoglycemia is achieved through maintaining an average blood glucoselevel of a subject above about 70 mg/dL, or above about 50-60 mg/dL.Preferably the blood glucose level of the subject is maintained underabout 140-200 and at least under about 350 mg/dL. Preferably, thesubject's blood glucose level is maintained so that normoglycemia ismaintained.

As will be apparent to one of skill in the art upon consideration of thedisclosure herein, any of the many different forms of insulin, as wellas any of the many different routes of administration of insulin,including those both approved by the FDA and in development, can be usedin the presently disclosed methods and formulations. Moreover, any ofthe currently available formulations of glucagon can similarly be usedin the methods and formulations. However, because glucagon has been,prior to the present disclosure, administered only parenterally tocontrol hypoglycemia, the present disclosure provides new glucagonderivatives, new formulations of glucagon and glucagon derivatives, andmethods of administering glucagon and glucagon derivatives that areparticularly suited to achieve the benefits provided by some of thepresent embodiments, including delayed and/or extended action glucagon.

While the precise dosage of insulin and glucagon will vary from patientto patient and depend upon a variety of factors, including but notlimited to age and sex of the patient, type and severity of diabetes,past history of the patient, including hypoglycemic and hyperglycemicepisodes, type of insulin and glucagon employed, and the like, thedosage for any patient can be determined, in light of the presentdisclosure, by one of skill in the art. The beneficial effects of someof the embodiments can generally be achieved by administering bothinsulin and glucagon in the ratio of about 1 unit of Insulin to about0.02-40 milliunits of glucagon (0.02 to 40 micrograms), when theglucagon is administered I.V. A unit of insulin is defined as the termis typically used for the treatment of diabetes, e.g. approximately 34.2micrograms to approximately 40 micrograms. The amount of insulin canalso be measured in international units (IU). A unit of glucagoncorresponds to 1 milligram of glucagon. In one embodiment, the ratio is1 unit of insulin to 0.2 to 4.0 milliunits of glucagon (0.2 to 4.0micrograms), when the glucagon is administered I.V. and the insulin isadministered s.c. When the glucagon is to be administeredsubcutaneously, 1 unit of insulin can be administered in the amount of0.02 to 200 milliunits of glucagon for each unit of insulin administeredor more than 40 to 200 milliunits of glucagon, e.g. 40 to 200 milliunitsper hour to a 100 kg person for each unit of insulin administered. Inanother embodiment, for each unit of insulin, 48-150 mU, 50-120 mU, or80-100 mU of glucagon is administered. In a preferred embodiment theglucagon is administered subcutaneously and the ratio is about 1 unit ofinsulin to more than 5 to about 20 ng/kg glucagon, which amount ofglucagon is administered each minute during the period of effectivenessof the insulin dose. In one embodiment, 1 unit of insulin isadministered, and the glucagon is administered at a rate of 8-12ng/kg/min. A standard dose can be created, for example, for treating a100 kg person for 1 hour in association with 1 unit of insulin. As willbe appreciated by one of skill in the art, this dose can be for basalinsulin rates. When postprandial levels of insulin are desired, theamount of glucagon in the dose will be increased accordingly. In someembodiments, the glucagon is administered subcutaneously in an amountbetween more than 5 ng/kg/min. and less than about 20 ng/kg/min. Morepreferably, the amount is between about 8 and 16 ng of glucagon/kg/minfor subcutaneous administration. Of course, one of skill in the art willappreciate that other dose equivalent amounts (i.e., the same effectiveamount administered through an alternative method) can also bedetermined in light of the teachings herein. As will be appreciated byone of skill in the art, and as shown in more detail below, this amountcan be adjusted to correspond to the amount of glucagon required toprevent hypoglycemia, without inducing hyperglycemia. Thus, in someembodiments, the amount of glucagon administered, even through s.c.administration, is less than the 5-20 ng/kg/min values described aseffective in the prevention of insulin-induced hypoglycemia. As will beappreciated by one of skill in the art, while the present disclosurefocuses on Type 1 diabetes, similar methods and compositions can be usedfor Type 2 diabetes. In general, the amount of glucagon can be increasedseveral fold over what is disclosed herein for Type 1 diabetes. Forexample, Type 2 diabetes can require 1.5 to 5 fold more glucagon, andpreferably involves two to three fold more glucagon than Type 1diabetes.

Any of the currently available forms of insulin, including but notlimited to recombinant human soluble (regular) insulin, human insulinanalogs, animal insulins, derived, for example, from beef, pork andother species, as well as delayed release forms, including intermediateand long acting insulin may be used for the herein disclosedcompositions and methods. Moreover, any of the currently used routes ofadministration, as well as newer routes in development, can be employed,including but not limited to subcutaneous, intramuscular, andintravenous injection, as well as oral, buccal, nasal, transdermal,sublingual, and pulmonary airway administration. Typical doses and doseranges for the administration of insulin to control diabetes known inthe art are suitable for use in the methods and compositions of some ofthe embodiments.

For example, prandial short-acting insulins, such as regular insulin andthe LISPRO, ASPART, and GLULISINE derivatives thereof, are well known inthe art and commonly used to treat diabetes. Such insulins can be usedto illustrate the embodiments in a manner applicable to other forms,including but not limited to NPH, LENTE, SEMI-LENTE, DETEMIR,ULTRA-LENTE, and GLARGINE (LANTUS), and pre-mixed formulations ofregular and long-acting insulins. In this illustration, the molecularweights ascribed to all three of these prandial short-acting insulinsare similar, with LISPRO at 5808, ASPART at 5825.8, GLULISINE at 5823and regular insulin at 5807. The molecular weight ascribed to glucagonis 3483.

The usual range of prandial insulin injections in Type 1 diabetes can beapproximated as two standard deviations from the mean, resulting in aninsulin dose range of 2-20 units. More than 95% of Type 1 diabetics willbe administered a prandial insulin dose within this range. The threeprandial insulins noted above all achieve peak serum concentrationswithin 1-2 hours after subcutaneous administration and have a durationof effectiveness of about 5 hours.

Currently, hypoglycemia is treated by a single parenteral injection ofglucagon in a dose of about 1 mg (1 unit); it has been determined thatthis dose is a gross excess of the dose actually required to controlhypoglycemia. When glucagon is given subcutaneously or intramuscularly,serum glucagon peaks within an hour, and its effects can persist forseveral hours. However, it appears that currently marketed forms ofglucagon are not stable in liquid form, either isolated or in vivo forprolonged periods of time, and in one embodiment, the present inventionprovides new pharmaceutical formulations of glucagon that are morestable, and new methods for using the stabler forms of glucagon that arecurrently available but not in widespread use.

It has been discovered, based in part on the respective times to peakserum level and durations of action of the prandial insulins andsubcutaneously administered glucagon, that there is a mismatch betweensubcutaneous insulin and glucagon pharmacokinetics. One embodiment ofthe present invention provides longer-acting glucagon formulations andderivatives that can be used to correct this mis-match, where desired orof benefit to the patient. “Longer-acting” glucagon, as used herein,refers to a glucagon that has a half-life greater than that of standardglucagon, including both natural extract and rDNA produced syntheticglucagon.

To provide the dose of glucagon required to achieve a duration of effectthat is similar to that of the prandial insulins, one can use a dosethat approximates the basal replacement dose. The usual basal glucagonreplacement dose by IV infusion is 0.5-0.75 ng/kg/min; one can assumethat a wider range of glucagon infusions, from as low as 0.10 to 5.00ng/kg/min (more often, 0.10 to 3.00 ng/kg/min) can be effective,depending on the patient, the insulin dose, the method of administration(e.g. I.V. vs. s.c.), and other factors. For example, in s.c.administration, the present inventors have discovered that the amount ofglucagon administered can be higher, as the bioavailability of glucagonadministered by subcutaneous infusion can be as low as 10%, and as lowas about 35% for bolus subcutaneous administration. Thus, the dose willbe increased or decreased accordingly to obtain the equivalenttherapeutic effect of administering glucagon at a rate of 5 to 20ng/kg/min. To match the PK of the insulins, these glucagon infusionrates would be continued for a period of time ranging from 150 minutesto 300 minutes. In some embodiments, the period of time of infusion canlast longer than 6 hours, for example 6-7, 7-10, 10-15, 15-20, 20-24hours, or longer. One can then multiply the replacement rates by theminimum and maximum times to give the total dose/kg. If one assumes thatthe typical Type 1 diabetic has a weight within the range of 50 to 100kg, and that the high and low range dose of subcutaneous prandialinsulin injection is between 2 and 20 units, then the insulin/glucagonratios can be calculated as shown in Table 1 below (showing the ratiosfor s.c. administration). Of course, this dosage replacement can happenthrough IV infusion of a dose equivalent amount. In one embodiment, thesame calculations described above are used to determine the amount ofdelayed or extended release forms of glucagon to administer to apatient, taking into account that there will be a lower level ofglucagon available initially and a higher amount of glucagon availablelater. While the amount of glucagon released at any point in time maynot be precisely known, enough glucagon is released per unit of timefrom the administered formulation so that, on average, about 0.1 to 5.0ng/kg/min. is released into the patient for embodiments in which theglucagon is administered through an I.V. In one embodiment, 0.5, 2, 3,or 4 times as much glucagon may be released on any given unit of time,depending on the patient, the type of diabetes being treated, and themode of administration.

In some embodiments, and as illustrated in table 1, the glucagon isadministered subcutaneously and is administered in an amount between 0.1to about 30 ng/kg/min., about 4.0 to about 20 ng/kg/min., about morethan 5.0 to about 30 ng/kg/min, about 6 to 25 ng/kg/min, about 6 to 20ng/kg/min, or about 8.0 to about 12.0 ng/kg/min.

TABLE 1 Insulin (sc)/Glucagon (sc) Weight Ratios (ng/ng) and InverseRatio [% terms] Patient Weight 50 kg 50 kg 100 kg 100 kg Duration ofGlue. Admin. 150 min 300 min 150 min 300 min 2 U Ins/6 ng/kg/min Gluc1.8 [56.3%] 0.9 [112.5%] 0.9 [112.5%] 0.4 [225%] 20 U Ins/12 ng/kg/min8.9 [11.3%] 4.4 [22.5%] 4.4 [22.5%] 2.2 [45%] Gluc 2 U Ins/18 ng/kg/min0.6 [168.8%] 0.3 [337.5%] 0.3 [337.5%] 0.1 [675%] Gluc 20 U Ins/6ng/kg/min 17.8 [5.6%] 8.9 [11.3%] 8.9 [11.3%] 4.4 [22.5%] Gluc 2 UIns/12 ng/kg/min 0.9 [112.5%] 0.4 [225%] 0.4 [225%] 0.2 [450%] Gluc 20U/18 ng/kg/min Gluc 5.9 [16.9%] 3.0 [33.8%] 3.0 [33.8%] 1.5 [67.5%]Explanation of Table Entries: For 2 U Ins/6 ng/kg/min Gluc: Means that aTOTAL of 2 Units of Insulin are administered over the given infusionperiod, and that Glucagon is administered over the period of infusion ata rate of 6 ng/kg/min. The two number in the Table given for a 50 kgperson over 150 minutes are the weight ratios of Insulin and Glucagon(absolute terms) and the weight ratios of Glucagon to Insulin inpercentage terms, (e.g., 1.8 = 80000 ng (2 units of Insulin)/750 ng (50× 150*6 Glucagon); 56.3% = 1/1.8 in percentage terms = inverse ratio).

In Table 1, the amount of glucagon administered ranges from 5.6 to 675%of the amount (in weight) of insulin administered; for many patients,however, the glucagon is administered at, or is present in a compositionat <225% of the weight of insulin. As will be appreciated by one ofskill in the art, the amount of glucagon administered can vary dependingupon many factors. Thus, ranges of the percent of glucagon to insulincan vary between 5.6 to 675%, e.g. more than 188% to less than 675%. Inone embodiment, the amount of glucagon administered is expressed as aratio to the amount of insulin administered; for example, the ratio ofglucagon administered can be from 5.6 to 11.3 percent of the amount ofinsulin administered. In some embodiments, the amount of glucagonadministered is 112 to 225% of the amount of insulin administered. Asshown in Example 7 below, these amounts of glucagon can induce elevatedblood glucose levels that approach hyperglycemia, as such, it is likelythat lower dose ranges can be sufficient to prevent hypoglycemia,without risking hyperglycemia. Lower ranges or doses can be from 0.09%to 188% of the weight of insulin, for example. As will be appreciated byone of skill in the art, the weight of the patient can vary, frominfants, e.g. 2 kg to full adults, e.g. 150 kg to 200 kg, or more.

In other embodiments, the amount of glucagon administered can bedescribed as an amount of glucagon by weight or activity independent ofthe amount of insulin administered; for example, in one embodiment, theamount of glucagon administered is 200-300 or 360-900 micrograms over anine hour period. In one embodiment, the amount of glucagon administeredis between about 22 to 33 micrograms in one hour. In one embodiment, theglucagon administered ranges from more than 5 ng/kg/min to about 30ng/kg/min s.c. In some embodiments, the amount is about 0.1 ng/kg/min toabout 30 ng/kg/min. In one embodiment, the amount is about 8 to about 16ng/kg/min, or about 12 ng/kg/min. s.c. In some embodiments,substantially lower values can be sufficient as well, depending upon thecircumstances and mode of administration.

In one embodiment, hypoglycemia is a blood glucose level of less thanabout 50-60, and generally less than about 70 mg/dL, and hyperglycemiais a blood glucose level more than about 140 to 200 mg/dL. In oneembodiment, excessive hyperglycemia is defined as a blood glucose levelabove 350 mg/dL. The ratio of glucagon to insulin and amounts of each isset, in accordance with the methods of the invention, to keep the bloodsugar level effectively between the hypoglycemic level and thehyperglycemia level. In another embodiment, the blood sugar level ismaintained between the hypoglycemic level and the excessivehyperglycemia level. In another embodiment, the blood sugar level ismaintained between the hyperglycemia level and the excessivehyperglycemia level. As will be appreciated by one of skill in the art,the level need not be observed precisely, and minor dips below or peaksabove these ranges are permissible. In one embodiment, the dose to beadministered to a patient is therapeutically equivalent to a dose of 0.5to 0.75 ng/kg/min of glucagon administered I.V. or is therapeuticallyequivalent to a dose of above 5 to about 20 ng/kg/min. s.c., i.e., 8-16ng/kg/min of glucagon via s.c. administration. In some embodiments, 0.1to 5 ng/kg/min. is all that is needed for the subcutaneousadministration, for the prevention of the onset of hypoglycemia. In someembodiments, the same amount of glucagon (or effective ratio of glucagonto insulin) is used, even if an agent other than insulin is used tolower or control blood sugar levels. Thus, in some embodiments, themethod of the invention may be practiced with an agent other thaninsulin, as the co-administration of glucagon, in light of the presentdisclosure, with a hypoglycemic agent is contemplated. Similarly, insome embodiments, a hyperglycemic agent other than glucagon is used toprevent the onset of hypoglycemia in insulin treated diabetics. In someembodiments, neither insulin nor glucagon is used, and a diabeticpatient is simultaneously administered both a hyperglycemic agent (anagent that causes blood sugar levels to rise) and a hypoglycemic agent(an agent that causes blood sugar levels to decline).

As will be appreciated by one of skill in the art in view of the instantdisclosure, the amount of glucagon or insulin administered to a patientcan vary depending upon the mode of administration For example, theamount of glucagon (or ratio of insulin to glucagon) to be added can bedescribed in terms of the amount to be administered via an I.V. (as inPCT Pub. No: WO 2004/060387, incorporated herein by reference). Thisamount can differ greatly depending upon how the glucagon is to beadministered, e.g. subcutaneously or via inhalation. For simplicity, theamount of glucagon required to achieve an equivalent result can bedescribed as a “dose equivalent.” For example, a “10 ng/kg/min. s.c.dose equivalent” is the amount required to achieve the same result aswould be achieved by administering 10 ng/kg/min. to a patientsubcutaneously. A “s.c. dose equivalent for I.V. administration” is theamount of glucagon administered intravenously that is required to obtainthe same amount of glucose or glucagon in the blood achieved bysubcutaneous administration of the amount of glucagon. Thus, in thelatter phraseology, the first method of administration describes whatthe dose administered is going to be an equivalent to using another modeof administration, and the second mode of administration recited is themode of administration actually employed. An I.V. dose equivalent ofglucagon administered subcutaneously will typically be more than theamount recited for I.V. administration, as can be seen by comparing thetable above to Table 1 in PCT Pub. No. WO 2004/060387. For example, aunit to be delivered I.V. is in some patients 0.1 ng/kg/min., while theamount for the same effect to be delivered subcutaneously can be 8ng/kg/min. in those patients. In addition, in a clinical trial describedin the Examples below, the amounts of glucagon required to beadministered to induce hyperglycemia in an insulin-treated diabetic werefor some patients in the 8-16 ng/kg/min. range (although lower doseswere seen to be effective as well), so the amounts of glucagon requiredmerely to prevent hypoglycemia will be below that range in somepatients. Given the present disclosure, one of skill in the art will beable to determine the appropriate amount in each circumstance.

As will be appreciated by one of skill in the art, the “amount ofglucagon administered” is not necessarily the amount of glucagon thatactually enters the bloodstream of a patient. Rather, for example,administering 9 ng/kg/min. s.c. of glucagon to a patient means that aninitial solution of an initial known amount was created, and based onthat amount, 9 ng/kg/min. of glucagon is administered to a patient. Ifthere is a loss or degradation of the glucagon prior to administration,then less glucagon enters the patient, and if there is a loss ordegradation of glucagon as it progresses to the patient's bloodstreamand tissues, the effective therapeutic dose is lower still. As will beappreciated by one of skill in the art, the actual amount of glucagonthat is active and enters the patient's circulation will in suchinstances be less than, in this example, 9 ng/kg/min.

One of skill in the art, given the present disclosure, can determine thedose equivalent for various methods or modes of administration. Thisdose equivalent can also vary depending on inter- and intrapatientvariability and the bioavailability of the drug. For example, if oneassumes glucagon administered through an I.V. is 100% bioavailable, thencertain glucagon formulations administered through a s.c. bolus can haveabout 35% bioavailability, while the same glucagon formulationadministered by continuous subcutaneous infusion can have abioavailability of 10%, as shown with patient data in the Examplesbelow. Additionally, differences between the method of administration ofinsulin and of glucagon can also be taken into account and determinedthrough the methods and examples provided herein. One way this can bedetermined is through various assays of insulin, glucose and glucagon ina patient following various routes of administration of the glucagon,e.g., as illustrated in Example 7.

The pharmaceutical compositions for use in many embodiments of theinvention can comprise those compositions useful in conventional methodsfor the control of diabetes and treatment of hypoglycemia. Suchconventional methods, as that phrase is used herein, include thoseapproved by the FDA, those in development, and those described inDiagnosis and Management of Type II Diabetes, by S. V. Edelman and R. R.Henry (5^(th) Ed. PCI Publishers), the entire text of which isincorporated herein by reference, and Chapters 7 and 8 of which areespecially pertinent. As used herein, a pharmaceutical formulation orpharmaceutical composition may contain a pharmaceutically acceptableexcipient, diluent or carrier. The phrase “pharmaceutically acceptable”means that the carrier, diluent or excipient is compatible with theother ingredients of the formulation and administration equipment andnot deleterious to the recipient thereof. Pharmaceutically acceptableexcipients are well known in the art. See, e.g., Remington: The Scienceand Practice of Pharmacy (19th edition, 1995, Gennavo, ed.).

In one embodiment, the glucagon or similar substance is administered ina buffer. Appropriate buffers are those that maintain the mixture at apH range from about 6.0 to about 9.0, but which do not interfere withthe function of glucagon. Examples of buffers include, but are notlimited to, Goode's buffers, HEPES, Tris, ammonium acetate, sodiumacetate, Bis-Tris, phosphate, citrate, arginine, histidine, and Trisacetate. The selection of one or more appropriate buffers is within theskill of one of ordinary skill in the art.

The control of diabetes by insulin therapy, as well as the control ofhypoglycemia by glucagon therapy, can involve parenteral administrationof the insulin or glucagon. Parenteral administration may be performedby subcutaneous or intramuscular injection by means of a syringe,optionally a pen-like syringe. Some of the embodiments of the methodscan be practiced using such methodology, although, as noted above, itmay be preferable in some instances to provide glucagon in a manner thatensures that its duration of action more closely matches that of theinsulin employed such that the glucagon is present when the risk ofhypoglycemia is greatest—typically a relatively long time after eatingbut still within the period in which the insulin administered continuesto exert its effect.

Where subcutaneous administration of insulin and glucagon are desired, avariety of methods may be employed to achieve the benefits of diabetescontrol and prevention of hypoglycemia. In one such method, a glucagonwith a shorter duration of action than the insulin is administeredwithin about one to four hours after the insulin is administered. Thismethod provides benefit in that most hypoglycemic episodes begin severalhours after the patient has last eaten, and many patients administerinsulin shortly before a meal. Thus, by delivering the glucagon a fewhours after the insulin, but prior to the onset of hypoglycemicsymptoms, one can achieve the benefits of the methods of the invention.Certain embodiments provide methods for controlling diabetes withreduced risk of inducing hypoglycemia by administering insulin incontinuation with a long acting glucagon and formulations thereof. Thus,in one embodiment, compositions having a long acting form of glucagonare provided.

In general, long acting forms are also known as extended release,prolonged release or controlled release (or similar term) forms. In oneembodiment, delayed or slow acting glucagon is a particular form of anextended or long release form of glucagon. Delayed acting glucagon iswithin the general class of long acting glucagon, as delayed or slowacting glucagon will allow for glucagon activity to occur after a periodof time following the administration of glucagon; however, delayedacting glucagon is effective in lower amounts at the initialadministration and increases in effectiveness over time. The glucagonitself may be long acting in nature, or it may be combined with othercomponents that allow its release over an extended amount of time.

In another embodiment, the insulin and glucagon can be administeredsimultaneously, with the insulin and optionally the glucagon deliveredparenterally, typically by subcutaneous injection. In this method, aglucagon with a longer duration of action is preferably employed, or theglucagon is administered by a route that provides a longer duration ofaction, e.g., as by continuous infusion, as illustrated in the Examplesbelow.

Such glucagon includes, but is not limited to, the glucagon, glucagonformulations, and routes of administration described in U.S. patentapplication publication No. 2002114829 and U.S. Pat. Nos. 6,197,333 and6,348,214, which describe liposome formulations of glucagon that providefor reduced dosage effect and are long acting; PCT patent publicationNo. WO0243566, which describes the delivery of glucagon via trans-dermalpatch; U.S. Pat. No. 5,445,832, which describes a long-acting glucagonformulation in polymeric microspheres; PCT patent publication No.WO0222154, which describes a slow-release glucagon that can have aduration of action measured in weeks; and U.S. Pat. No. 3,897,551 andGreat Britain U.S. Pat. No. 1,363,954, which describes the prolongationof glucagon duration by iodination (each of these publications areincorporated herein by reference in their entireties.) In an embodiment,the glucagon is administered as a slow-release or depot formulation(e.g., comprising polyethylene glycol).

There are many techniques known to those skilled in the art formodifying the release and/or pharmacokinetic characteristics ofproteins, include the modification of the amino acid sequence at thesite corresponding to the metabolic deactivation point associated withthe protein. These techniques and compositions include, “pegylation” orPEG-modification of the protein (see, for example, PCT Patentpublication Nos. WO0232957, WO9831383, and WO9724440, EP patentpublication Nos. EP0816381 and EP0442724, and U.S. Patent PublicationNo. 2002/0115592; U.S. Pat. Nos. 5,234,903; and 6,284,727); otherpolymer encapsulations (see EP patent publication No. EP0684044);lipophilic modification (see U.S. Patent Publication Nos. 5,359,030;6,239,107; 5,869,602; and 2001/0016643; EP patent publication No.EP1264837; and PCT Patent publication Nos. WO9808871 and WO9943708;formulating into liposomes (see U.S. Pat. Nos. 6,348,214 and 6,197,333);serum albumin modification (discussed in more detail below and in PCTPatent publication Nos. WO02066511 and WO0246227 and U.S. Pat. No.4,492,684); formulating in the form of emulsions, microspheres,microemulsions, nanoencapsulation and microbeads (see U.S. Pat. Nos.4,492,684; 5,445,832; 6,191,105; 6,217,893; 5,643,604; 5,643,607; and5,637,568); formulations involving ligands (see PCT Patent publicationNo. WO0222154); and iodination (see U.S. Pat. No. 3,897,551).

In one embodiment, an iodination method of increasing half life (asdescribed in U.S. Pat. No. 3,897,551; see form I3G) is employed.Iodinated glucagon has extended activity (measured in terms of elevatedglucose levels) of between 1 and 3 hours, depending on the extent ofiodination. In one embodiment LISPRO insulin and I3Glucagon are admixedso that the modified glucagon is present at approximately 1.5% by weightof the insulin in the mixture (keeping the concentration of insulin perml in the LISPRO formulation constant). Because of the longer lastingeffect of the modified glucagon, a smaller proportion of glucagon toinsulin by weight will be required to prevent hypoglycemia in somepatients.

Another form of a long acting glucagon is a zinc-protamine-glucagonformulation. Examples of such Zinc protamine-glucagon formulations areknown in the art (See, for example, Kaindl et al., Verh Dtsch Ges InnMed. 1972; 78:1099-101; Kaindl and Kuhn, Z Gesamte Inn Med. 1972 Dec.15; 27(24):1097-8; Christiansen and Tonnensen, Med Scand. 1974 December;196(6):495-6; Gamba et al., Minerva Med. 1977 Nov. 3; 68(53):3613-26;Kollee et al., Arch Dis Child. 1978 May; 53(5):422-4; Kalima andLempinen, Ann Chir Gynaecol. 1980; 69(6):293-5; Aynsley-Green et al.,Arch Dis Child. 1981 July; 56(7):496-508; Schmid and Wietholter, DtschMed Wochenschr. 1982 Nov. 26; 107(47):1809-11; Day and Mastaglia, Aust NZ J Med. 1985 December; 15(6):748-50; Cederblad et al., Horm Res. 1998;50(2):94-8; all herein incorporated in their entirities by reference.

Additionally, Pichler et al. (Wien Klin Wochenschr 19:91(2):49-51(1979)) demonstrated that a zinc protamine form of glucagon had amaximal effects up to 3 hours after the actual administration of thedrug, and only displayed a decrease in activity after the fourth hour.Thus, the effective half-life of zinc protamine glucagon is in the rangeof hours.

In one embodiment, glucagon is combined with zinc without protamine, asdescribed in Tarding et al., (European Journal of Pharmacology,7:206-210 (1969), hereby incorporated in its entirety by reference).This also results in a long acting form of glucagon. In one embodiment,the mixture involves a 1 to 2 ratio of zinc to glucagon.

In one embodiment, the zinc protamine glucagon is made in a mannersimilar to how zinc protamine insulin is made, apart from thereplacement of insulin with glucagon. In one embodiment, zinc glucagonand zinc protamine glucagon is made as described in Tarding et al.(European Journal of Pharmacology 7:206-210 (1969)). For example, zincglucagon can be made by suspending freeze-dried zinc glucagon crystalsin a zinc acetate buffer, for a final concentration of 1 mg glucagon/ml,0.05 mg zinc/ml. Alternatively, the zinc protamine glucagon can beprepared by suspending freeze-dried zinc glucagon crystals in a zincacetate buffer containing protamine to a final concentration of 2 mgglucagon/ml, 0.15 mg zinc/ml, and 0.5 mg protamine/ml.

Another example of an agent that can be included with glucagon incompositions useful in the present methods includes a protamine sulfate,as described in combination with GLP-1 in U.S. Pat. No. 6,703,365,(issued Mar. 4, 2004, to Galloway et al.). The GLP-1 combination thereindisclosed displays an increased half-life and an increased shelf life aswell. Any glucagon which displays an increased half-life can be usefulin the present methods and compositions.

Another means by which the half-life of the protein may be extended isthrough the use of “serum binders”, such as can be achieved through theconjugation of albumin to glucagon by a connector. In one embodiment,the glucagon contains a moiety which allows it to attach itself toalbumin in vivo. Alternatively, the glucagon may be modified such thatit is able to connect to a connector, which will then allow the glucagonmolecule to be associated with a protein such as albumin in vivo. Thus,the modified glucagon can be directly added to a patient, where it willsubsequently bind to albumin in the host, which will in turn result inthe extension of the useful life of glucagon in the system. Thisapproach has been described for other purposes for GLP-1 in U.S. PatentPublication 20030108568, published Jun. 12, 2003 to Bridon et al., aswell as for various other proteins (See, for example, U.S. Pat. Nos.6,277,863, issued Aug. 21, 2001 to Krantz et al., 6,500,918, issued Dec.31, 2002, to Ezrin et al., 6,107,489, issued Aug. 22, 2000, to Krantz etal., 6,329,336, issued Dec. 11, 2001, to Bridon et al., and 6,103,233issued Aug. 15, 2000, to Pouletty et al., all of which are incorporatedby reference in their entireties). In one embodiment, the bindingbetween glucagon and albumin occurs with the aid of biotin and avidin orstreptavidin. In another embodiment, glucagon can be attached to otherproteins through the use of maleimide groups and sulfur groups. Theglucagon can be attached to any suitable protein, not only albumin.

In one embodiment, a prolonged release form of glucagon is a gel orfibril based form of glucagon. These may be prepared as described inGratzer and Davies (European J. Biochem., 11:37-42 (1969), herebyincorporated in its entirety by reference.)

Other forms of prolonged release glucagon are also contemplated for usein the present methods. Long release preparations may be made usingpolymers to complex or absorb the glucagon. The controlled delivery maybe exercised by selecting appropriate macromolecules and theconcentration of macromolecules as well as the methods of incorporationto control release. For example, diffusion controlled systems may beused. Examples of such materials include particles of a polymericmaterial such as polyesters, polyamino acids, polyvinylpyrrolidone,methylcellulose, carboxymethylcellulose, hydrogels, poly (lactic acid),or ethylene vinylacetate copolymers. Alternatively, instead ofincorporating a compound with these polymeric particles, it is possibleto entrap a compound of some of the embodiments in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules, respectively, or in colloidal drug deliverysystems, for example, liposomes, albumin microspheres, microemulsions,nanoparticles, and nanocapsules, or in macroemulsions. Such teachingsare disclosed in Remington's Pharmaceutical Sciences (1980). In oneembodiment, the rate of dissolution of the drug is primarily controlledby the dissolution of a shell, which encapsulates the drug.

In one embodiment, an osmotic pump system is used to provide a prolongedrelease of glucagon, allowing the rate of release of the drug to becontrolled by the inflow of water across a semipermeable membrane into areservoir that has an osmotic agent. In another embodiment, ion exchangeresins are used to control the release of glucagon.

In one embodiment the extended release form of glucagon is apreproglucagon [Lund, et al., Proc. Natl. Acad. Sci. U.S.A. 79:345-349(1982)]. This polypeptide is subsequently processed to form proglucagon,which is subsequently cleaved into glucagon and a second polypeptide(Patzelt, C., et al., Nature, 282:260-266 (1979)). Thus, byadministering this form of prepro- or proglucagon, one is able to delaythe onset of the activity of the glucagon.

In one embodiment, glucagon is administered in a form that allowssubstantially no release of active glucagon initially, and then allowsfor a small amount of release of glucagon over time. Such a form ofglucagon is useful when prolonged periods between drug intake will occurand the desired result is an effect towards the end of the prolongedperiod, for example, during nocturnal periods. The form may be inherentin the glucagon protein itself, i.e., a semi-synthetic glucagon variant,due to compositions associated with the protein, due to the formulationsin which the glucagon is administered, due to the route and method bywhich the glucagon is administered, as well as other reasons asdescribed herein.

Glucagon with a low activity level is desirable in some circumstances.As the delivery of precise amounts of small volumes can be difficult,especially over prolonged periods of time, compositions of glucagoncomprising components that lower the activity of glucagon may bedesirable in some situations to allow the administration of largervolumes of sample. Alternatively, variants or mutants of glucagon with alower activity level can be used to achieve this result. The term“glucagon” can encompass both wild-type glucagon and variants orderivatives of glucagon.

In some embodiments, a combination of an insulin component and a slowrelease form of a glucagon provided by the invention is employed in themethods of the invention. As will be appreciated by one of skill in theart, the combination may be achieved through a single or multipleformulations and single or multiple means of administering the insulincomponent or the glucagon component.

In one embodiment, parenteral administration is performed by means of aninfusion pump. A variety of insulin pumps are available and in commonuse that are suitable for delivery of the insulin and glucagoncompositions (as well as suitable for the delivery of insulin, withglucagon being delivered by another route, such as transdermal). Suchpumps include, for example and without limitation, the pumps marketed byMedtronic (such as the MiniMed), Animas Corporation, Disetronic, andDana. The glucagon can optionally be administered with the insulin, anda glucagon with a short duration of action can be employed, as theglucagon can be administered as necessary. In one embodiment, theglucagon can be administered intravenously at a rate of 0.5-0.75ng/kg/min or within the wider range of about 0.10-5 ng/kg/min,alternatively, within the range about 0.10-3 ng/kg/min. or in an amountthat is an intravenous dose equivalent. In a preferred embodiment, theglucagon is administered subcutaneously and is administered in an amountbetween about 4 and 30 ng/kg/min, about more than 5.0 up to 25ng/kg/min, about 8.0 to 20 ng/kg/min, or about 8.0 to 12.0 ng/kg/min.Lower amounts of glucagon can be administered (0.1-5 or 2-5 ng/kg/min.)subcutaneously to prevent hypoglycemia in some patients. In oneembodiment a dose will prevent hypoglycemia without causing excessivehyperglycemia. Hyperglycemia is a blood glucose above the normal range.Glucagon can elevate blood glucose above where it would be without theadministration of exogenous glucagon, and in a preferred embodiment, thedose administered is one that is still protective against hypoglycemiabut only minimally elevates blood glucose above the levels maintained inthe patient when not suffering from hypoglycemia.

Thus, in one embodiment, the present invention provides a new drugdelivery device, a pump suitable for the delivery of insulin for thecontrol of diabetes, and for the delivery of glucagon for the control ofhypoglycemia in a human, i.e., the pump contains both insulin andglucagon. The pump may include a reservoir containing both insulin andglucagon. In other embodiments, the pump includes insulin and glucagonin two separately controlled reservoirs. A method of controllingdiabetes in a human patient to reduce the risk of hypoglycemia isprovided, said method comprising administering both insulin and glucagonto the diabetic patient using a pump of one of the embodiments describedabove.

In another method, either the insulin or the glucagon or both isprovided in a formulation that is a powder or a liquid suitable foradministration as a nasal or pulmonary spray or for ocularadministration. A variety of such formulations are known for insulin andglucagon, and the present disclosure provides methods for using theseknown formulations for administering either one independently, as wellas for administering the corresponding formulations of the embodimentsthat comprise both insulin and glucagon to control diabetes with areduced risk of inducing hypoglycemia.

Methods and formulations for nasal, pulmonary, or ocular administrationinclude those in PCT patent publication Nos. WO0182874 and WO0182981,which describe aerosolized insulin and glucagon; European patentpublication EP1224929 and U.S. Pat. No. 6,004,574, which describe aninhaled glucagon with melezitose diluent; U.S. Pat. No. 5,942,242, whichdescribes formulations of insulin and formulations of glucagon suitablefor nasal administration; U.S. Pat. No. 5,661,130, which describesformulations suitable for ocular, nasal and nasolacrimal or inhalationroutes of administration; U.S. Pat. No. 5,693,608, which describesmethods and formulations for the nasal administration of insulin and forglucagon; U.S. Pat. No. 5,428,006, which describes methods andformulations for the nasal and other mucosal administration of insulinand for glucagon; U.S. Pat. No. 5,397,771, which describes methods andformulations for the mucosal administration of insulin and of glucagon;U.S. Pat. No. 5,283,236, which describes methods and formulations forthe ocular administration of insulin and of glucagon; and Europeanpatent publication EP0272097, which describes a formulation of glucagonfor nasal administration. In addition to these formulations, the methodsof delivering these formulations as described are also contemplated.

In one embodiment, compositions and methods are provided for controllingdiabetes with a reduced risk of inducing hypoglycemia by administeringinsulin and glucagon, in which one or both of the insulin and glucagonis administered transdermally, e.g. from a patch, optionally aiontophoretic patch, or transmucosally, e.g. bucally. Manufacture anduse of transdermal delivery devices are well known in the art (see,e.g., U.S. Pat. Nos. 4,943,435 and 4,839,174; and patent publication no.US 2001033858). The transdermal delivery of glucagon, and a patentpublication describing transdermal formulations of glucagon, has beencited above, and U.S. Pat. No. 5,707,641 describes methods andformulations for the transdermal delivery of insulin.

Moreover, some embodiments of the methods can be practiced by oraladministration of both insulin and/or glucagon in the therapeuticallyeffective amounts and their dose equivalents described herein. Methodsand formulations for the oral administration of insulin and of glucagoninclude those described in PCT patent publication No. WO9703688.

The insulin and/or glucagon employed in the methods and formulations canbe supplemented with or replaced by compounds and compositions that havesimilar activities or effects. For example, glucagon may be replacedwith glucagon mimetics or variants of glucagon.

Insulin can be replaced or supplemented with hypoglycemic agents,including but not limited to Insulin Sensitizers, DPP IV inhibitors, andGLP1 analogs, insulin secretagogues including, but not limited to,sulfonylureas such as Acetohexamide (DYMELOR), Chlorpropamide(DIABINESE), Tolazamide (TOLINASE), Tolbutamide (ORINASE), Glimepiride(AMARYL), Glipizide (GLUCOTROL), Glipizide Extended Release (GLUCOTROLXL), Glyburide (DIABETA, MICRONASE), Glyburide Micronized (GLYNASE,PRESTAB); Meglitinides such as Nateglinide (STARLIX) and Repaglinide(PRANDIN); Gastric Inhibitory Polypeptide (GIP); Glucagon-like peptide(GLP)-1; Morphilinoguanide BTS 67582; Phosphodiesterase inhibitors; andsuccinate ester derivatives; insulin receptor activators; insulinsensitizing Biguanides such as Metformin (GLUCOPHAGE),Thiazolidinediones (TZD) such as Troglitazone (REZULIN), Pioglitazone(ACTOS), and Roziglitazone (AVANDIA); Non-TZD peroxisome proliferatoractivated receptor γ (PPARγ) agonist GL262570; Alpha-glucosidaseinhibitors such as Acarbose (PRECOSE) and Miglitol (GLYSET); Combinationagents such as Glucovance (GLUCOPHAGE with GLYBURIDE); TyrosinePhosphatase Inhibitors such as Vanadium, PTP-1B inhibitors, and AMPKactivators, including 5-aminoimidazole-4-carboxamide ribonucleoside(AICAR); and other agents such as Exendin (EXENATIDE (syntheticexendin-4)) and Amylin (SYMLIN® (pramlintide acetate)),D-Chiro-Inositol, altered peptide ligands (NBI-6024), Anergix DBcomplex, GABA inhibit melanocortin, Glucose lowering agent (ALT-4037),Aerodose (Aerogen), insulin mimics, Insulin-like growth factor-1 aloneor in a complex with BP3 (SOMATOKLINE), metoclopramide HCL (Emitasol/SPD425), motillde/Erythromycin analogs, and GAG mimetics.

In one embodiment, variants of glucagon are contemplated. Such variantsmay comprise a single or many amino acid changes, for example, from oneto all of the amino acids may be changed, relative to the native humanglucagon sequence, so long as the resulting variant functions asrequired herein. In one embodiment, the HELIX content is adjusted at theC-terminus, as well as partial agonists are combined to provide more ofa “basal” input. In one embodiment, transient PEG-modifications at theN-terminus, to control activation can be complemented with mutationsintroduced at the C-terminus, for controlling affinity. Examples of suchvariant glucagon molecules, and their resulting characteristics andactivities, are available in the art. For example, Sturm et al., (J.Med. Chem. 41:2693-2700 (1998)) teaches a general structural functionrelationship for some of the salt bridges in glucagon. In oneembodiment, the variant glucagon has a Lys substitution at positions 17and 18, and a Glu substitution at position 21, resulting in a variantwith a 500 percent binding affinity and a 700 percent relative potency.In one embodiment, the variant glucagon amide has only a Lyssubstitution at position 17, resulting in a variant with a 220 percentbinding affinity and a 230 percent potency. In another embodiment, thevariant glucagon amide has a Nle substitution at position 17, a Lyssubstitution at position 18, and a Glu substitution at position 21,resulting in a variant with 150 percent binding affinity and a 300percent potency. In another embodiment, the variant glucagon moleculesdisplay low binding ability or low activity. Thus, for example, aglucagon amide variant with a lysine at position 18 may be used, as itonly has 36 percent of the normal binding affinity and only 12 percentof the normal potency. Another example would be a glucagon variant witha Phe at position 18, which has only 4.7 percent of the normal bindingaffinity and 0.9 percent of the relative potency.

In one embodiment the only pharmaceutically active components of theformulation are insulin and glucagon. In one embodiment, thepharmaceutical composition (e.g., containing both insulin and glucagon)is not formulated as an aerosol and/or does not contain troglitazonehydrochloride (and may not contain any thiazolidinedione). In anembodiment, the formulation is not administered orally and/or is notadministered nasally. In one embodiment, the pharmaceutically activecomponents of the formulation are administered transdermally, but notthrough a patch. For example, the active components can be administeredthrough the use of a cream.

As discussed above, the simultaneous effective administration of lowdoses of glucagon together with insulin can help prevent insulin-inducedhypoglycemic events. The prevention of these events will have variousbeneficial results.

Repeated mild to moderate hypoglycemic events can result in a loss ofhypoglycemic awareness by the subject. Thus, the subject may no longerbe able to detect that he or she is actually experiencing a hypoglycemicevent, increasing the risk that more hypoglycemic events can occur.Thus, the above compositions and methods can be optimized so as toreduce the risk of this occurring. The above ratios of insulin andglucagon and amounts of glucagon recited for the prevention ofhypoglycemia can be sufficient to treat this condition, and the methodsof the invention include methods for adjusting the amount administeredto achieve the desired therapeutic effect for a particular patient, modeof delivery, or formulation. In some embodiments, the combination ofglucagon and insulin is administered to a patient to prevent the loss ofhypoglycemic awareness by the subject. In other embodiments, theglucagon and insulin are administered so as to restore hypoglycemicawareness to the subject. This can be achieved by administering anamount of glucagon so that additional episodes of hypoglycemia arereduced or prevented. The amount can vary, e.g. 8-16 ng/kg/min.administered subcutaneously or 0.1-5 ng/kg/min intravenously.

As will be appreciated by one of skill in the art, these therapies andcompositions can be useful not only for people with diabetes but withanyone taking insulin or other hypoglycemia inducing agent.

As will be appreciated by one of skill in the art, not every episode ofmild or moderate hypoglycemia needs to be prevented. The amount orpercent of events inhibited can vary by the particular situation andsubject and can include inhibiting 2-5, 5-10, 10-20, 20-30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, 90-99, or 99 percent to all of themild/moderate hypoglycemic events. Additionally, hypoglycemia need notbe prevented in every case and can be delayed in some embodiments. Anyamount of delay can be useful, for example, a delay of 1-10, 10-30,30-60, 60-120, 120-300, 300-600 or more minutes. Alternatively, a delayof an additional 1-20, 20-60, 60-100, 100-200, or 200% to 10 foldAdditionally, not all of a patient's sensitivity to hypoglycemia needsto be restored or preserved, e.g. 1-10, 10-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, 90-99, 99-100% can be restored or preventedfrom loss. “Hypoglycemic sensitivity” or “hypoglycemia unawareness” canbe based on the individual's ability to detect the occurrence of ahypoglycemic event. For example, hypoglycemia unawareness can be aninability to detect 1-20, 20-40, 40-50, 50-70, or more percent of thehypoglycemic events (e.g., glucose levels fall below 50 mg/dL in theblood). Alternatively, the inability to detect a particular symptom ofhypoglycemia can also be used to determine hypoglycemia unawareness andhow successfully it is being treated. Signs and symptoms include, forexample, shakiness, dizziness, sweating, hunger, headache, irritability,pale skin color, sudden moodiness or behaviour changes, clumsy or jerkymovements, difficulty paying attention, confusion, and a tinglingsensation around the mouth. A description of various possible categoriesof hypoglycemia can be found in “Defining and Reporting Hypoglycemia inDiabetes” Diabetes Care, 28:1245-1249 (2005), hereby incorporated byreference in its entirety. In particular, symptomatic, asymptomatic, andprobably symptomatic hypoglycemia involve plasma glucose levels below orequal to 70 mg/dl. As noted herein, in some situations, lower levels ofblood glucose can also be used as a threshold.

Kits

In some embodiments, the compositions described herein are provided inkit form. In one embodiment, the kit comprises a vial of glucagon, avial of insulin, a means for administration, such as a syringe or pump,and instructions for the administration of the glucagon and insulin. Insome embodiments, the glucagon and insulin are premixed in a singlevial. In other embodiments, the insulin and glucagon are premixed in asyringe. The particular instructions will vary depending upon thedesired use of the kit, e.g. for nocturnal control of hypoglycemia orotherwise. The instructions can be determined by one of skill in theart, given the present disclosure and the particular use intended forthe kit. In one embodiment, the instructions will describe the methodsdisclosed herein.

Exemplary kits contain glucagon and one or more of the followingpackaged together: (1) insulin; (2) a solution (e.g., excipient) forresuspending or diluting glucagon (3) a device for administeringglucagon and/or insulin; and (4) instructions. In one embodiment, thedevice (3) contains the glucagon and/or contains insulin.

A kit may comprise glucagon in a powder form within a sterile vial witha standard septum seal. In one embodiment, the vial contains a mixtureof 1 mg of lyophilized glucagon, 49 mg lactose, and hydrochloric acid toadjust the pH (glucagon is soluble below pH 3 or above pH 9.5). The kitalso has a pre-filled glycerine syringe, which contains 12 mg/ml ofglycerine in a mixture of water, and hydrochloric acid. A secondcontainer holds a 1 mg/mL solution of insulin, which may be stored inliquid form in a syringe. The kit further has instructions, instructingthe user to inject 1 mL of diluent from the pre-filled glycerine syringeinto the vial. The instructions then direct the user to collect anamount of the glucagon/glycerine solution into the syringe containingthe insulin. This amount will vary depending upon intended use and theparticular user and may be determined by a physician.

In one embodiment, the volume of glucagon collected in the syringe isbetween 0-5% of the volume of insulin to be injected. The kit maycomprise tables and/or charts allowing for ease of use and customizationto determine what amount or ratios should be used for each user andsituation.

The entire dose in the insulin syringe can then be injected (childrenare typically administered 50% of a standard dose, and the kit can bemodified accordingly). In one embodiment, the insulin syringe and theglycerine syringe are one and the same, in which case the startingamount of glucagon is lower to maintain the appropriate ratio ofglucagon to insulin that is injected. In another embodiment, theinsulin, glucagon and glycerine are premixed in the kit. Instructionsare adjusted accordingly for the particular embodiment used. In oneembodiment, the kit comprises a glucagon kit, an insulin kit, andinstructions for how to combine the two kits. As will be appreciated byone of skill in the art, any of the above discussed compositions ormethods may be included in the kit as components or instructions. Thus,for example, various methods of administration, various compositions ofinsulin or glucagon, and various buffers or solvents may be used in thekits. In one embodiment, a means of administering insulin rapidly iscombined with a means of administering glucagon more slowly. In oneembodiment, the kit comprises only a form of glucagon with a set ofinstructions.

In one embodiment, the instructions can direct the user to administermore than 5 to 20, e.g. 6 to 16, ng/kg/min. of glucagon subcutaneously.In one embodiment, the instructions will direct the user to administer30-45 ng/kg/hour of glucagon. In one embodiment, the units of glucagonare in 1500 ng and 2250 ng size doses for a 50 kg person, one dose to betaken each hour. In another embodiment, the units of glucagon are in3000 to 4500 ng size doses, for a 100 kg person, one to be taken eachhour. The kit can include a device for subcutaneous administration. Inone embodiment, the units of glucagon are in a 36 microgram size dosefor a 50 kg person, one dose to be taken each hour. In anotherembodiment, the units of glucagon are in 24 to 96 or 36 to 96 microgramsize doses, for a 100 kg person, one to be taken each hour. Thesesubcutaneous values can be sufficient to create hyperglycemia in somediabetic patients; thus, in some embodiments, less glucagon is required,e.g. an amount similar to that to be administered intravenously, 0.1-5ng/kg/min. or higher. In some embodiments, the kits include instructionsregarding doses for the age, weight, and sex of the individual. In oneembodiment the instructions include information concerning doses to takein view of future activities, such as sleeping, eating (e.g., how muchand what type of food), sitting, or exercising. As will be appreciatedby one of skill in the art, an I.V. or s.c. dose equivalent can also beused if glucagon is to be administered in another manner. In someembodiments, the kits contain unit doses of glucagon to be added withthe insulin. For example, a unit dose can be about 50 or 100 micrograms(or milliunits), which can be sufficient to protect a 100 kg subject fora one hour period from hypoglycemia. Unit doses can be prepared for 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24 or more hoursor days. Smaller doses for smaller people or fractional hours are alsocontemplated.

Unit Doses of Glucagon

In one embodiment, rather than providing a mixture of glucagon andinsulin, unit doses of glucagon alone are provided so as to be readilyadministrable to a subject as required, to prevent insulin-inducedhypoglycemia. As the ratio of glucagon to insulin can be low, the amountof glucagon in the unit dose can also be low. As will be appreciated byone of skill in the art, the actual amount of glucagon in each dose willdepend upon the characteristics of the individual, possible activitiesthat the individual is going to do or has done, how the dose is to beadministered and the form of the glucagon. Thus, the doses describedbelow are only representative of some of the possible doses. The dosagecan be determined by the skilled practitioner in view of the presentdisclosure.

A unit dosage form of glucagon contains a discrete quantity of glucagonfor administration, and may be in the format of tablets, capsules, orpowders in a container such as a vial or ampoule, cartridge, syringe,inhaler, transdermal patch, or other container or package. The quantityof glucagon in a single unit dose form preparation is typically from0.002 mg to 0.1 mg. However, as will be appreciated by one of skill inthe art, the amount in each dose can vary based on the manner that thematerial is to be administered. The previous units are for I.V.administration. For s.c. administration, the amount of glucagon in aunit dose can be, for example, from 0.002 mg to 0.2 mg or 0.036 mg to0.4 mg for a 100 kg person, which dose should be effective for at least1 hour. Preferably, the unit dose is between about 40 micrograms and 300micrograms, and more preferably, between about 50 and 100 micrograms. Ofcourse, these numbers can be adjusted based on the size of the averageperson to be treated and the duration of hypoglycemia prevention desiredper unit dose. For example, with slow release formulations, it can beparticularly advantageous to include sufficient glucagon for releaseover 2-3, 3-5, 5-8, or more hours. Thus, larger doses are possible, incertain circumstances. Lower amounts, even through s.c. administration,can also be used to ensure that hyperglycemia does not even transientlyoccur.

A single unit dosage form contains sufficient glucagon for a singleadministration of glucagon as described herein. Unit dosages can bedesigned for particular events. For example, they can be designed foruse before or after the administration of insulin. Alternatively, theycan be designed for administration in view of activities such as eatingor exercising or going to sleep. As the amount of glucagon to beadministered will depend upon various factors of a patient, such aslifestyle and weight, the unit dose can be expressed in universal unitsfor ease of adjusting the dose. Additionally, how the unit is to beadministered can also alter the amount of glucagon one places in eachunit dose. These universal unit dosages are actually unit doses that aredivided into smaller individual parts. Thus, a 50 kg individual can take5 parts of these universal unit doses, while a 100 kg individual mighttake 10 parts. This allows greater customization of the glucagon intake.Of course, lower amounts of glucagon, e.g. similar to I.V.administration, can also be used if lower levels of elevated blood sugarare satisfactory. Thus, even for subcutaneous administration, a unitdose can be between 0.036 mg and 0.2 mg or 0.002 mg and 0.2 mg, forexample.

In a related aspect, a pharmaceutical preparation of glucagon in dailyunit dosage form is provided. The daily dosage form contains sufficientglucagon for one day, including the case in which glucagon isadministered multiple times during a single day as described herein. Forexample, there may be multiple (e.g., 2 or 3 or more) glucagonadministrations after a meal or meals during the day (see, e.g., Example1 and/or administrations for prevention of nocturnal hypoglycemia; orcontinuous administration via, for example, transdermal patch or pump).In one embodiment, for example, via I.V. administration, 0.02 mg to 1 mgglucagon is provided in a container accompanied by instructions (e.g., alabel) that the glucagon should be administered as separate doses overthe course of a day. Usually the amount of glucagon is not more thanabout 0.04 U. and is often not more than about 0.02 U. not more thanabout 0.01 U. not more than about 0.005 U and sometimes not more thanabout 0.002 U. In one embodiment, the amount of glucagon given over oneday is about 0.84 mg via I.V. or a dose equivalent for s.c. injection.For subcutaneous administration, 960 to 4800 micrograms of glucagon canbe provided for administration over a day. In one embodiment, theglucagon is in a slow release form that is given all at once. In anotherembodiment it is in the form of, for example, 6 pills, one to be takenevery four hours.

In a related aspect, a pharmaceutical preparation of glucagon inmultiple dosage form is provided. A multiple dosage form of glucagon cancontain a sufficient dose for administration for one, two, three, four,five, or six days, one week, or even more than one week. In oneembodiment, for example, 0.02 to 0.036 mg to 1 mg of glucagon isprovided in a container accompanied by instructions (e.g., a label) thatthe glucagon should be administered as separate doses over the course ofa day or more than one day.

In one embodiment, a daily dose or multiple dose of glucagon is preparedby resuspending a powder in a liquid excipient, and a portion of theresulting solution can be administered at each administration during theday (or several days in the case of some multiple dose forms).

In addition to glucagon, the unit dosage form, daily dosage form ormultiple dosage form can include other components, such as excipients,buffers, stabilizers, carriers and the like, as well as otherpharmaceutically active agents. In one embodiment, as discussed above,the unit dose includes insulin or an insulin secretagogue.

Multiple doses of glucagon (e.g., multiple daily doses) can be packagedtogether in a box, bubble-wrap, or in other well known formats.

In general, the dosage forms will be labeled or will be accompanied byinstructions for proper dosing. For example, the daily dosage form maybe labeled to indicate the number and/or weight or volume of unit dosesin the container. The dosage forms may also be labeled or otherwiseindicate the age of the patient for whom the preparation is intended.For example, the dosage form may be indicated as suitable for adults,children over 15 years of age, children over 10 years of age, childrenover 5 years of age, and the like.

In one embodiment, any of the above glucagon dosages may comprise aextended release glucagon. In such embodiments, the dosage isappropriately adjusted, as disclosed herein, to maintain a blood glucoselevel within the desired ranges.

Glucagon Solutions

Administration of low doses of glucagon (e.g., less than 0.01 U) can beinconvenient using formulations prepared according to conventionalmethods (e.g., resulting in an approximately 1 mg/ml solution).Accordingly, in some embodiments, a lower concentration glucagonsolution is made and/or administered. Administration, as used in thiscontext, includes self-administration (whether by injection, by infusionusing a pump, or other methods) and administration by another. Invarious embodiments, glucagon is administered as a solution having aconcentration of less than about 0.25 mg/ml, for example, less thanabout 0.2 mg/ml, less than about 0.1 mg/ml, less than about 0.05 mg/ml,less than about 0.01 mg/ml, or even less than about 0.005 mg/ml ofglucagon. In an embodiment, the concentration of glucagon is preferablyat least about 0.001 mg/ml. In another embodiment, the concentration ofglucagon is at least about 0.01 mg/ml. Such amounts can be appropriatefor I.V. administration and dose equivalent amounts can be created forother methods of administration. For example, if the method ofadministration is s.c. injection, then the concentration of glucagon canbe higher, at least about 0.01 mg/ml, or 0.05 mg/ml, or, 0.2 mg/ml, 0.5mg/ml, or between about 0.5 mg/ml and 2 mg/ml of glucagon. In someembodiments, these doses are combined with a device that can administerthe doses in low amounts over a prolonged period of time, such as apump.

Glucagon can be resuspended in any pharmaceutically acceptable carrier,diluent or excipient.

In one embodiment, a pharmaceutically acceptable formulation of glucagoncontains a concentration of less than about 0.25 mg/ml, less than about0.2 mg/ml, less than about 0.1 mg/ml, less than about 0.05 mg/ml, lessthan about 0.01 mg/ml, or even less than about 0.005 mg/ml when it is tobe administered I.V., or a dose equivalent amount for other methods ofadministration. For example, for compositions that are to beadministered subcutaneously, a pharmaceutically acceptable formulationof glucagon contains between 0.5 and 2 mg/ml of glucagon.

In a related embodiment, a method of preparing a glucagon formulationfor therapeutic use is provided and involves adding an aqueous solutionto a composition comprising glucagon (such as, but not limited to, asingle unit dose, daily dose or multiple doses of glucagon as describedabove) in a quantity that results in a solution containing glucagon at aconcentration of less than about 0.25 mg/ml, less than about 0.2 mg/ml,less than about 0.1 mg/ml, less than about 0.05 mg/ml, less than about0.1 mg/ml, or even less than about 0.05 mg/ml for I.V. administration.Concentrations 2 to 10 fold higher (or more) can be used for othermethods of administration, such as subcutaneous administration. Thesolution can contain other agents, both pharmaceutically active and/orinactive.

In one embodiment, the glucagon solution is loaded into, or is containedin, a device for delivery to a patient. In some embodiments, the devicecontains at least about 0.1 ml, at least about 0.2 ml, at least about0.3 ml, at least about 0.4 ml, at least about 0.5 ml or more than 0.5 mlof a glucagon solution.

In a related aspect, the further step of administration orself-administration to a human subject with diabetes is provided. In oneembodiment, the subject does not exhibit symptoms of hypoglycemia. Inone embodiment the human is an adult. In one embodiment the human isolder than 10 years old, and optionally older than 15 years old orolder. In one embodiment, the subject is not suffering from a stomachailment.

In one embodiment, any of the above glucagon solutions may comprise anextended release glucagon. In such embodiments, the dosage isappropriately adjusted, as disclosed herein, to maintain a blood glucoselevel within the desired ranges described herein.

The methods and compositions disclosed herein may be used to treat humanpatients as well as other mammals (e.g. rats, mice, pigs, non-humanprimates, and others). In some embodiments the human patient is a childor juvenile; in one embodiment the human patient is an adult. In someembodiments the patient is a Type I diabetic. In one embodiment thepatient is a Type II diabetic. In one embodiment the patient is abrittle Type I or Type II diabetic. In one embodiment, the non-humanmammal is an animal model for the study of diabetes, e.g. Zuckerdiabetic-fatty (ZDF) rats, and db/db mice.

While many of the examples and much of the description provided hereinis explicitly directed to subcutaneous administration of low doses ofglucagon, other aspects are also disclosed. For example, while thesubcutaneous doses described herein are generally greater than 5 andless than 20 ng/kg/min., other doses are also contemplated for thevarious embodiments described herein. For example, doses from 0.1 to 5ng/kg/min. for I.V. or s.c. administration, especially in combinationwith long acting forms of glucagon (such as zinc protamine), variouskits, and unit doses are contemplated along with the more than 5 to 20or 30 ng/kg/min. doses. Additionally, as pointed out below, doses at 4ng/kg/min., and lower, of s.c. glucagon can also be effective inpreventing or delaying hypoglycemia. Thus, in some embodiments, the doseadministered subcutaneously to prevent or delay hypoglycemia is about0.1-5, 1, 1-2, 2-3, 3-4, 4-5, more than 5, 5-6, 6-8, 8-12, 12-16, 16-20,or 20-30 ng/kg/min. Corresponding amounts for unit doses, other dosesfor administration, or kits are also contemplated. For example, a 1 hourunit doses of glucagon for a 100 kg person at 4 ng/kg/min. would contain24 micrograms of glucagon, and a 1 hour pharmaceutical composition wouldcontain 24 micrograms of glucagon and 1-3 units of insulin. As will beappreciated by one of skill in the art, any of the disclosed doses canbe turned into one hour unit doses or other aspects described hereingiven the present disclosure. This can depend upon dose amount (e.g.,0.1 to 30 ng/kg/min. or 6 to 16 ng/kg/min.), presence and amount ofinsulin (e.g. 0 or 2-20 Units), the size of the patient (e.g. 3-200 kg),and the number of hours of desired effectiveness (e.g., 0.5-24 or more).

The following examples describe illustrative embodiments of theinvention and are not intended to be limiting in any manner.

EXAMPLE 1 Co-Administration of Glucagon Parenterally and Insulin for theControl of Diabetes and Prevention of Hypoglycemia

The glucagon currently available in the North American market is humanglucagon of rDNA origin produced either by Eli Lilly & Co or BedfordLabs (Novo). Four brand names are known: Glucagon Diagnostic Kit(Lilly); Glucagon Emergency Kit (Lilly); Glucagon Emergency Kit for LowBlood Sugar (Lilly); and GLUCAGEN (Bedford Labs).

Novo produces glucagon under its own name outside of North America. Novoproduces its glucagon in yeast, and Lilly produces its glucagon in E.coli. The following examples illustrate practice of some of the methodsusing such commercially available glucagons and insulins administeredvia a variety of routes.

The Lilly glucagon is typically provided in kit form. The glucagonwithin the kit is in the form of a powder within a sterile vial with astandard rubber-sealed neck. The vial contains a mixture of 1 mg oflyophilized glucagon, 49 mg lactose, and hydrochloric acid to adjust thepH (glucagon is soluble below pH 3 or above pH 9.5). The patient injects1 ml of diluent from a pre-filled syringe (which contains 12 mg/ml ofglycerine in a mixture of water, and hydrochloric acid) into the vial.The vial is shaken until the solution is clear. The liquid is returnedto the syringe, and the entire dose is injected (children are typicallyadministered 50% of the standard dose).

Glucagon is administered parenterally by subcutaneous, intramuscular,and intravenous routes, with the pharmacokinetic properties differingaccordingly as understood by those of skill in the art. Maximum plasmaconcentration is achieved approximately 20 minutes after subcutaneousadministration. The half life in vivo ranges from 8 to 18 minutes. Peakplasma concentration of approximately 8 ng/ml are achieved afterapproximately 20 minutes, and elevated glucose levels persist forapproximately 1½ hours after administration and begin rising almostimmediately following administration. Patients with insulin-induced comawill typically recover consciousness within 15 minutes of glucagonadministration. Parenteral glucagon, when given to treat hypoglycemia,does so primarily by increasing serum glucose availability throughincreased output of glucose by the liver (the conversion of glycogen toglucose and formation of new glucose by gluconeogenesis).

There are a wide variety of insulin dosage regimes in use. The regimeused depends on whether Type 1 or Type 2 diabetes is being treated andon a large number of factors specific to the individual being treated.It is normal medical practice to replace insulin using a combination ofparenterally administered insulins (usually subcutaneously) of rapidonset/short acting duration (LISPRO (HUMALOG) or ASPART (NOVOLOG)),slower onset/short acting duration (regular human insulin), intermediateduration (NPH or LENTE), long acting duration (ULTRALENTE), BASULIN(Bristol Myers Squibb) and 24 hour peak-less duration (GLARGINE (LANTUS)and DETEMIR).

The dosage regimes can be quite complex. For example, a typicaltwice-daily regimen might involve administering short acting andintermediate duration insulin before breakfast and supper. The insulinprofile thus obtained has a number of peaks, which roughly correspond tothe anticipated post-prandial glucose output, as well as providing abasal insulin level throughout any 24 hour period. This is illustratedin FIG. 1, which shows the idealized pharmacokinetics for a mixture ofregular and intermediate acting insulin. The graph shows the effect of atwice-daily insulin regimen: Twice-daily administration of regular(solid lines) and intermediate-acting LENTE or NPH (dashed lines)insulins before breakfast and the evening meal provides peaks of insulinafter the injections as well as a relatively constant baseline level ofinsulin throughout the day after injections of the intermediate-actinginsulins.

Insulin levels can vary significantly between individuals and evenwithin the same individual, depending on factors such as site and depthof injection, local blood flow, total volume and type of insulininjection, and other factors appreciated by those of skill in the art.Thus, there can be significant inter and intra-patient variability insubcutaneous absorption of insulin, which increases the likelihood ofvariations in serum glucose, including the possibility of hypoglycemia.

With the advent of the rapid onset insulins and a long acting insulinwith little or no peak appearance GLARGINE (LANTUS); also, DETEMIR is along acting insulin in development; in addition, ULTRALENTE is a longacting insulin but tends to have some peak effect in most patients), itbecame possible to manage insulin levels (and hence blood glucoselevels) with more accuracy. The basic methodology is to replace basalinsulin and prandial insulin through the combined use of insulinpreparations having different rates of onset and durations of action.This may involve the use of separately administered insulins ofdiffering onset, (e.g. GLARGINE and LISPRO) or the use of variouspre-mixed formulations (e.g. 70/30-70% NPH and 30% regular combined),which are commercially available for this purpose.

The point at which the glucagon is administered is before, during, orimmediately prior to the period when insulin action is most unopposed,for example when significant insulin action persists in the absence ofsufficient serum glucose availability. Thus, insulin-inducedhypoglycemia may occur whenever there is a mismatch between circulatinginsulin and glucose levels (a relative excess of insulin effect toglucose availability).

Insulin Administered Parenterally

(i) GLARGINE/LISPRO/ASPART/GLULISINE Insulins

For this illustrative example, the patient (all patients referred toherein are fictitious, except for any reference to patients in theexamples describing actual clinical testing; any resemblance to anactual person is coincidental) is an adult male, 50 years of age,weighing 75 kg, with 5 L of blood, suffering from type-2 diabetes andusing insulin therapy (without concomitant oral combination therapy). Hehas been using insulin for over 10 years and his glucagon response tohypoglycemia is minimal. H is insulin regimen involves basal insulinreplacement using GLARGINE (LANTUS) subcutaneous injections at a dosagelevel of 20 units administered at bedtime in addition to prandialinsulin injections of LISPRO (HUMALOG), ASPART (NOVOLOG), GLULISINE(APIDRA) of between 5 and 10 units (depending on the amount ofcarbohydrate consumed) administered at mealtimes.

His insulin profile is very simple, being a flat line (basal level setby the GLARGINE (LANTUS)) punctuated by peaks corresponding to theprandial LISPRO (HUMALOG), ASPART (NOVOLOG), GLULISINE (APIDRA) insulininjections. This insulin profile is shown in FIG. 2. In this patient,the risk of hypoglycemia typically arises between 2 and 5 hours afterthe meal. It is during this period that administering glucagon is mostefficacious and effective in preventing the possibility of ahypoglycemic episode. In non-diabetics, glucagon usually falls followinga carbohydrate meal (in response to increased glucose levels) and thenrecovers subsequently as glucose levels return to normal. In type-1diabetics (and type-2 diabetics of 5 years or more) the glucagonresponse to low serum glucose is limited. Hence, if insulin causes theserum glucose level to drop well below the basal level, hypoglycemiawill ensue.

Hypoglycemic symptoms are typically observed in diabetics at glucoselevels less than 50 mg/dl, or sometimes less than 40 mg/dl. In normalindividuals, glucagon release would be augmented (by about 40 pg/ml orhigher, e.g. augmented by about 60 pg/ml or higher) before glucoselevels fell this low and would prevent the onset of hypoglycemicsymptoms. However, glucagon production (or regulation) is deficient inmany insulin-treated diabetics. Thus, administering glucagon to achievethese levels during the period of susceptibility will prevent orattenuate the severity of hypoglycemic attack.

The s.c. dose of glucagon required to provide prophylaxis is in someembodiments about 6-20 ng/kg/min. for basal insulin levels, and aproportionally higher amount for higher levels of insulin.

Thus, in accordance with the methods, this patient is administered 41 to90 micrograms of glucagon subcutaneously after the meal. Two similardoses are administered hourly for an additional two hours. This makesthree doses at hours 2, 3, and 4, providing protection from hypoglycemiafor 3 hours beginning 2 hours after the meal. With formulationscomprising both insulin and glucagon, one can use a lower percentage ofglucagon (5.6%), as the risk and degree of hypoglycemia is (in part)insulin-dose-dependent. In this example, while the glucagonconcentration from the dose administered 2 hours after the meal willhave fallen back to approximately basal levels after an hour, theelevation of blood glucose due to this dose will persist for more thanone hour, giving time for the second dose to take effect. The samepharmacokinetics applies to the third dose of glucagon. In analternative embodiment, two doses or even one dose of glucagon can beadministered.

Although this example employs a simple basal and prandial insulin model,it will be understood by those skilled in the art to be applicable toall currently practiced dosage regimens. The timing (and frequency) ofthe glucagon injections may be adjusted to match the period in which thepatient is most susceptible to hypoglycemia, i.e. the point at whichinsulin action and glucose availability are most mismatched.

(ii) NPH/Human Insulins

A typical diabetic patient is an adult male, 63 years of age, weighing75 kg, suffering from Type 2 diabetes for 18 years and using combinationinsulin therapy (without concomitant oral anti-diabetic therapy). In thepast, he will typically have used oral anti-diabetic medications,including Glyburide and Glipizide, but these will have been stopped andinsulin started when his serum glucose levels were consistently above250 mg/dl. He will have been using insulin of one type or another forover 10 years and will have developed evidence of backgroundretinopathy, mild renal impairment with a serum creatinine of 1.9 mg/dland creatinine clearance of 60 ml/min, mild proteinuria, bilateraldistal symmetrical neuropathy in both feet, and exertional angina. Hisinsulin regimen would typically involve a split-mixed regimen ofsubcutaneous NPH insulin, 20 units before breakfast and 15 units beforedinner, which is intended to provide day-long basal insulin coverageplus modest postprandial coverage for lunch and the evening meal (andbedtime snack). In addition, he injects regular insulin of between 6 and10 units (depending on the level of pre-meal serum glucose as well asthe size and carbohydrate content of the meal) before these meals.

His insulin profile is similar to that shown in FIG. 1, with less rapidpeaks and slower decays resulting from the prandial injections ofregular insulin and slower onset and delayed decay effects from thetwice daily intermediate acting NPH insulin. His fasting glucose levelsare typically well controlled in the range of 90-130 mg/dl, but h is 1-2hour postprandial glucose levels are suboptimal and generally range from180 to 240 mg/dl. Glycosylated hemoglobin is elevated at 7.9% (normalrange 4-6%). Efforts to increase his breakfast or evening meal dose ofprandial regular insulin to reduce postprandial glucose levels isusually accompanied by frequent intermittent hypoglycemia, of mild tomoderate severity, often 1-2 hours before lunch or several hours afterdinner. These episodes of hypoglycemia can be quite severe andassociated with symptoms of sweating, tremors, nausea, and headaches,particularly when he is late for meals. He has never had insulin-inducedhypoglycemic coma but is reluctant to increase his insulin dosage incase this happens. He fears that he could lose his driver's license ifthis occurs or perhaps his job as a night watchman. Because of thispatient's long history of diabetes and presence of significantcomplications, it is expected that he will exhibit impaired glucosecounter-regulation to hypoglycemia, especially manifest as a blunted orabsent glucagon response.

In this patient, the risk of hypoglycemia is usually greatest between 3and 5 hours after a meal (late postprandial hypoglycemia), whencirculating insulin levels are still increased above fasting level butglucose availability (from gastrointestinal absorption and liverproduction) is minimal. It is during this period, prior to the onset ofhypoglycemia, that the administration of glucagon would be mostefficacious and effective in preventing the possibility of hypoglycemicepisodes by increasing circulating glucose availability. In non-diabeticindividuals, both insulin and glucagon are tightly regulated following ameal to balance glucose production and utilization so as to maintainnormoglycemia. Should insulin effects become pronounced, glucagon levelswill rise to offset this hypoglycemic potential.

To prevent the development of hypoglycemia in accordance with oneembodiment of a method, this patient is administered 36-96 milliunits ofglucagon, administered subcutaneously (optionally using Eli Lilly'sGlucagon Emergency Kit, as described above) two to three hours aftereach meal. In one embodiment, the glucagon is added when glucosemeasurements indicate glucose levels are approaching hypoglycemiclevels. This administration provides the required protection betweenhours 3 and 5, as described above.

Although this illustrative example employs a simple basal and prandialinsulin model, it will be understood by those skilled in the art to beapplicable to virtually all currently practiced insulin dosage regimens.The glucagon injections are optimally timed and vary depending on theinsulin regimen used but are designed to achieve sustained glucagonlevels during the expected periods of relatively unopposed insulinaction.

In the current hypothetical example, this situation tends to occur atseveral times throughout the day. For example, hypoglycemia is prone tooccur when the “tail” of injected regular insulin absorption combineswith peaking insulin availability from the intermediate acting NPH. Thissituation occurs several hours after breakfast when serum glucoseavailability (primarily from gut absorption and liver production) isminimal or decreasing. Similar situations also often occur beforedinner, at bedtime, and in the middle of the night. Thus, for allinsulin dosage regimens, the timing of glucagon injection can varydepending upon the pharmacologic characteristics and timing of theinsulin(s) used.

To offset the glucose-raising potential of the administered glucagon,the dose of insulin acting during that hypoglycemic period can beincreased somewhat to maintain euglycemia. However, the increasedavailability of glucagon provides a buffering action or cushion to theexcessive glucose lowering action of insulin in those specificcircumstances as described above and blunts or prevents hypoglycemia.

B. Insulin Administered by Pump

In this example, the patient described in Example 1.A.i uses a pump toadminister his insulin requirement. Instead of administering basalinsulin by GLARGINE (LANTUS) once daily as in Example 1.A.i, thepatient's insulin pump is programmed to provide a continuous stream ofrapid-onset insulin (e.g. LISPRO or ASPART). In this example, headministers ASPART in doses of between 5 and 10 units at mealtimesaccording to the pre-meal glucose level and the amount of carbohydrateand calories consumed. The patient will then, in accordance with thismethod, administer glucagon (using Bedford Lab's GLUCAGEN product, forexample) two hours after the meal and repeat the dose hourly for anothertwo hours. This amount can be about 36 to 96 micrograms of glucagon,delivered s.c. The glucagon is administered subcutaneously. Thisadministration provides protection from hypoglycemia between hours 2 and5, as described in Example 1.A.i. As will be appreciated by one of skillin the art, the glucagon can also be administered via a pump. In analternative example, the amount of glucagon is scaled up per unit ofinsulin; thus, 36-96 micrograms are used per unit of insulin.

C. Insulin Administered Transdermally [Including Patch and TopicalCream]

In this example, the patient is a 62 year old, lean Type 2 diabetic of 6years duration. He was initially treated with Glyburide 20 mg twicedaily and subsequently with the addition of Metformin 1 gram twicedaily, but fasting and postprandial blood glucoses were consistently inthe range of 200-350 mg/dl. He is advised by his physician that insulinis required. The oral anti-diabetic medications are discontinued andGLARGINE (LANTUS) insulin 15 units is administered at bedtime to providehis day-long basal insulin replacement needs. Postprandial insulin isadministered by transdermal patch to provide 2-6 units of rapidly actinginsulin (patches available in 2 unit increments; although this examplerefers to use of a patch, those of skill in the art will appreciate thatsubstantially similar methodology can be employed to practice theembodiment with insulin or glucagon delivered transdermally by othermeans, such as creams or lotions). Alternately, he is offered the24-hour basal insulin replacement patch instead of once daily GLARGINE.The basal insulin replacement patch contains insulin in a uniqueformulation designed to provide steady continuous absorption and lowconstant serum insulin levels throughout the day. Because of persistentelevation of fasting plasma glucose, his physician progressivelyincreases his dose of GLARGINE insulin over 6 months to 24 units andtransdermal patches to 4-10 units. With this increase in GLARGINE andtransdermal insulin dosage, fasting glucose levels ranged from 70-110mg/dl and 1-2 hour postprandial glucose levels from 130-180 mg/dl within3 months.

The patient applies the rapidly-acting insulin patches 30-60 minutesprior to meals. This timing is chosen so that absorption of the mealcoincides with insulin patch absorption kinetics and action. Thispatient has near normal glycemic control as indicated above but beginsto suffer from early morning hypoglycemia, typically at 1 or 2 a.m. Atthese times, this hypothetical patient is frequently confused,irritable, and at times anxious. Several readings of finger-stickglucose taken during these events reveal blood glucose values of 35-40mg/dl with prompt resolution of symptoms with ingestion of juice. In aneffort to control these bouts of hyperglycemia, his physician graduallydecreases the evening dose of GLARGINE, but this is associated withdeterioration in glycemic control and, primarily, elevation ofpre-prandial glucose levels.

To restore near-normal glycemia but prevent early morning hypoglycemicsymptoms in accordance with some embodiments of the methods, thephysician increases the GLARGINE insulin back to 24 units at bedtime andprescribes administration of subcutaneous glucagon at 18 ng/kg/min. ofintended protection (using Bedford Labs Glucagon product) immediatelyfollowing the injection of GLARGINE at ˜23:00. The time ofadministration of glucagon depends primarily on the rate of absorption,which is rapid, reaching peak levels within 15-30 minutes, and aduration of action of approximately 2-3 hours. In one embodiment, plasmaglucagon approximating “high normal basal levels” is maintained duringthis period and prevents an unopposed action of insulin from GLARGINEinsulin or a delayed action of the early evening (pre-dinner) patch. Forexample, glucose levels of more than 120-160 mg/dl are contemplated. Inan alternative embodiment, the low end of the normal glycemic levels areset as a goal for the blood glucose level. This therapy provides therequired protection from hypoglycemia for approximately 3 hours afterthe GLARGINE injection, as described above. With the addition of bedtimeglucagon to his diabetes regimen, the early morning hypoglycemicepisodes should resolve and day-long near-normal glycemia be preserved.As will be appreciated by one of skill in the art, a s.c. or i.v. doseequivalent of glucagon can also be administered in the same manner asthe insulin (e.g., transdermally via patch or cream).

D. Insulin by Inhalation [Including Pulmonary, Buccal Nasal andSublingual]

This example is similar to Example 1.A.i, except the patient administersinsulin by inhalation rather than by subcutaneous injection. It will beunderstood by those skilled in the art that similar methods apply wheninsulin is administered buccally, nasally, or sublingually in accordancewith these methods, although a dose equivalent amount will be applied.The patient will either continue to administer his basal need viaGLARGINE (LANTUS) or he will utilize an insulin inhaler to administerbasal insulin needs. The patient will administer his prandial insulinneed (equivalent to between 5 and 10 units administered by subcutaneousadministration) using his insulin inhaler (either pulmonary, nasally,buccally, or sublingually).

In accordance with these methods, the patient will then subcutaneouslyadminister 45 micrograms of glucagon (optionally using Lilly's Glucagonkit) two hours after the meal and another two doses hourly thereafter.He will administer the glucagon subcutaneously. This will provide therequired protection from hypoglycemia between hours 2 and 5, asdescribed in Example 1.A.i. As will be appreciated by one of skill inthe art, the glucagon can also be administered via inhalation, in a doseequivalent amount. Alternatively, a lesser amount of glucagon isadministered, for example, 3-5 micrograms of glucagon for 0.5 to 2.0units of insulin. As will be appreciated by one of skill in the art, theprecise amount of glucagon administered can vary and can be determinedfor various amounts of insulin, via the method shown in Example 8 below.

EXAMPLE 2 Co-Administration of Glucagon by Pump and Insulin for theControl of Diabetes and Prevention of Hypoglycemia

In one method, insulin can be administered by pump. There are a numberof pumps commercially available (or soon to be available) in the USmarket and elsewhere that are suitable for use in the present methods.These include but are not limited to:

-   -   ANIMAS (IR-1000 & IR-1200)    -   DELTEC (Cozmo pump)    -   DISETRONIC (H-TRONplus and D-TRONplus)    -   LIFESCAN & DEBIOTECH (MEMS Insulin Pump in development)    -   MEDTRONIC MINIMED (PARADIGM Insulin Pump and 508 Insulin Pump)    -   MEDTRONIC MINIMED (2007 Implantable Insulin Pump System (EU        only))

When both the insulin and the glucagon are to be administered by pump(from separate reservoirs), a number of configurations can be employedin the practice of the present method. Typical configurations are:

-   -   (1) A single device with a single pump and two reservoirs (for        dual reservoir pumps, see, for example, U.S. Pat. No. 5,474,552)        with each drug delivered through 2 separate lines that are        merged prior to cannulization;    -   (2) A single device with a single pump and two reservoirs with        each drug delivered through 2 separate lines, each of which is        independently cannulized;    -   (3) A single device with two independent pumps and two        reservoirs with each drug delivered through 2 lines that are        merged prior to cannulization;    -   (4) A single device with two independent pumps and two        reservoirs with each drug delivered through 2 separate lines,        each of which is independently cannulized;    -   (5) Two devices, each with a single pump and one reservoir, with        each drug delivered through 2 separate lines that are merged        prior to cannulization; and    -   (6) Two devices, each with a single pump and one reservoir, with        each drug delivered through 2 separate lines independently        cannulized.

It will be understood by those skilled in the art that otherconfigurations are possible and that the practice of the embodiments arenot limited to the devices and device configurations listed above. Forexample, implantable pumps may be used in almost exactly the same way asis achieved using external pumps.

Embodiment (1) above minimizes trauma to the patient on cannulization,reduces cost, simplifies infusions, and minimizes complexity. With thisembodiment, the single pump can be programmed to deliver appropriatevolumes from each reservoir, each containing different concentrations ofone of the two hormones. An example of such a pump is provided herein.

In a typical insulin pump, the internal pump mechanism usually comprisesan electromagnetically driven pulsatile pump having a solenoid operatedpiston mounted for reciprocation within a cylinder to draw medicationfrom an internal storage chamber (reservoir), and to deliver suchmedication through the delivery line and then via a cannula ormicro-cannula to the patient.

Because delivery lines used with pump insulin are typically one-half toone meter in length with lumen diameters of the order of 1/10th of amillimeter (dead volume of the order of a 1/10th of a milliliter orabout 10 IU of Insulin), the time delay between a new drug reaching thebody and the time at which the pump starts infusing it is likely to besubstantial (about half a day).

To reduce this delay, one embodiment provides pumps with lines of muchshorter length and/or of very small internal lumen diameter that enablethe lag time between a switch in drugs to be much shorter. The presentembodiment also provides peristaltic type pumps acting on two deliverylines.

One embodiment provides a system comprising a pump and a set of fourvalves, two immediately before and two immediately after the pump, whichwhen operated in pairs, control which drug is pumped. The two lines are,in one embodiment, merged at the point of cannulization, therebyeliminating the lag or (dead volume) time. The extra space required forthe electronically actuated micro-valves is minimal and adds little bulkor expense and can be assembled using commercially available devices.Additional possibilities involving the use of fewer than 4 valves aredescribed below.

In one embodiment, an economical pump system suitable for use in some ofthe methods is a micro-pump known as MEMS (Micro-Electro-MechanicalSystem), being developed for diabetes by Debiotech under the brand nameChronojet. The use of two such micro-pumps in a unitary device addslittle bulk and only minimal expense to existing designs. As notedabove, the two delivery lines can be merged (in the sense that the twodrugs come into direct contact) at the point at which they connect tothe cannula or similar micro-needle device used to puncture the skin anddeliver the drug.

In one embodiment, a single split-lumen (dual lumen) line is usedinstead of two physically separate lines. This method has the advantagethat the patient has only to route one flexible delivery line ratherthan two. Alternatively, two standard lines physically adhered alongtheir lengths can be used in accordance with some embodiments of themethods to achieve the same advantage.

In one embodiment, therefore, the pump is a currently used insulin pumpmodified to have two drug reservoirs instead of one, each beingindependently administered by a single (or dual) pump and a singlecontrol system to manage the quantity and relative timing ofadministration of the two drugs. As noted above, the device can in someembodiments comprise 1, 2 or 4 valves and appropriate connective tubing.Schematics of devices that can be used in accordance with some of themethods are shown in FIGS. 3, 4, and 5. In FIG. 3, the insulin reservoir(1) and the glucagon reservoir (2) have lines in fluid communicationwith pump (3) and then on to the cannula (5) via split lumen deliveryline (4) by way of 4 valves (6) and (7). When valves (6) are open andvalves (7) are closed, only insulin is pumped. When valves (6) areclosed and valves (7) are open, only glucagon is pumped. In this way, asingle pump may be used to deliver insulin or glucagon to the patient,either simultaneously or separately, with minimal mixing of the twosubstances by virtue of delivery through a split lumen delivery line inwhich the liquids only mix at the cannula, i.e. the point of deliver. InFIG. 4, the insulin reservoir (1) and the glucagon reservoir (2) havelines in fluid communication with pump (3) and then on to the cannula(5) via split lumen delivery line (4) by way of 2 valves (6) and (7).These are 2-way valves that allow either the insulin path or theglucagon path to be open—but only one at a time. A small amount ofmixing of the two fluids will take place in the small stretch of linethrough the pump, but this will be an insignificant volume when comparedto typical pumping volumes. In this way, glucagon and insulin may bedelivered to the patient with minimal mixing and dead space in thelines. The advantage of this construction over the constructiondescribed disclosed by FIG. 3 is that only two valves are required foroperation. In FIG. 5, the configuration here is the same as thatdisclosed in FIG. 4, but in this case the two valves (6) and (7) arecombined into a single device with a unitary actuation mechanism. Inthis way the mechanism is kept as cheap and simple as possible, withonly one valve and one pump required to achieve the desired result.

It is normal practice with pumped insulin for the patient to set thepump to deliver a basal level of insulin and to intervene manually toadminister prandial insulin as required.

In one embodiment, the glucagon delivery is automatically administeredover the appropriate period (for example continuously between the 3rdand 5th hours following the manual instruction to deliver the prandialinsulin). The control logic required to produce such a sequence ofevents can be programmed into the pump.

A. Insulin Administered by Pump

This example illustrates how some of the methods can be practiced usingpump-based administration. A typical hypothetical patient is an adultmale, 35 years of age, weighing 75 kg, having type-1 diabetes since theage of 15 and using insulin therapy from the time of diagnosis. He hasbeen on a number of different insulin regimens previously with less thanoptimal glycemic control. In the last 5 years, he has begun to developsignificant background retinopathy, mild renal insufficiency, andhypertension, and is concerned that these complications will continue toprogress rapidly unless he is able to improve glycemic control from hiscurrent glycosylated hemoglobin level of 7.8%. Most recently, he hasbeen on ULTRALENTE, 22 units, at bedtime and LISPRO insulin, 4-8 units,just prior to each meal and snack. The dose of ULTRALENTE has beenadjusted to provide basal replacement of insulin, while the dose ofprandial LISPRO varies depending upon the prevailing pre-injection serumglucose and total calories and carbohydrate content of each meal.

Despite self-monitoring of capillary blood glucose by glucometer 4-6times per day, his glycemic control is often erratic, ranging from highvalues in the 200 mg/dl range to occasional hypoglycemia. In theprevious year, he has had 3 bouts of severe hypoglycemia with coma ornear coma, two occurring while at work and the other following a game ofhandball. All required the assistance of others and in the post-exercisecase required the intramuscular injection of glucagon by paramedics.

With using insulin pump therapy, short acting insulin is typically used,because the pump provides a continuous feed to simulate basal insulinover the long term. In this example, the patient uses ASPART (NOVOLOG)as his insulin of choice. The pump, reservoir, and control mechanism (inthis illustrative case, the MEDTRONIC MINIMED PARADIGM INSULIN PUMP) canbe attached at various sites about the body of the patient (mostcommonly on the belt, for example), and is linked by a flexible plastictube to the micro-cannula he has inserted in his abdomen, thigh, or arm(women tend to place the infusion site in the lower abdomen while menusually choose the upper abdomen).

The patient has programmed the device to deliver 1 IU of insulin every50 minutes (20 units a day). When the patient eats during the day, heprograms the device to release an amount of insulin (between 3 and 8units) appropriate to his meal situation (pre-meal glucose, totalcalories, and carbohydrate content). He does this by pushing theappropriate buttons on the device (or by use of a remote control device,if available) to select the size of the insulin bolus required.

Following use of the programmable insulin infusion pump, the patient hasbeen able to achieve a marked improvement of glycemic control, withpre-prandial glucose levels ranging from 70-110 mg/dl, postprandial (1-2hour) levels of 120-160 mg/dl, and glycosylated hemoglobin of 6.4%.However, he continues to be plagued by frequent mild-to-moderatehypoglycemic episodes that he frequently doesn't recognize until hemeasures his fingerstick glucose. Many of these low glucose values arein the range of 30-40 mg/dl. He has been told by his wife and friendsthat at times he behaves inappropriately but improves with ingestion offood or juice.

Because of the long duration of type-1 diabetes and frequent and oftenunrecognized hypoglycemia, this patient has significant impairment ofglucose counter-regulation with absent glucagon and markedly bluntedepinephrine response to hypoglycemia. That is, he is unable to mount aneffective response to abnormally low blood glucose and the attendantdangers that can result. Furthermore, he has hypoglycemic unawareness,which frequently accompanies recurrent hypoglycemia and increases therisk of severe hypoglycemia developing. He is unaware when his bloodglucose is dangerously low, because his body's mechanisms to recognizelow blood glucose are defective. This is a common scenario in diabetesof long duration and manifests itself most commonly when efforts toachieve normal or near-normal glycemic control are attempted in suchindividuals. Because of his concern about the increasing frequency andseverity of his hypoglycemic episodes and his frequent inability torecognize them, he is seriously considering “loosening up” his glycemiccontrol to reduce the hypoglycemia. He understands that this may havedetrimental consequences with increased microvascular complications butfeels that the dangers of severe hypoglycemia are greater and moreimmediate.

This hypothetical patient has been striving to achieve the best glycemiccontrol possible based on the understanding that the risk of developingmicrovascular complications is minimised so long as his day-longglycemia approaches non-diabetic levels. Despite being on the mostadvanced and flexible form of insulin delivery system currentlyavailable and having achieved significant improvement in glycemiccontrol to recommended goals, he is plagued by frequent and potentiallydangerous bouts of hypoglycemia. To alleviate this situation, yet allowhim to maintain the same level of glycemic control, the methods providedherein are used, and in one embodiment, a second pump device, which canbe identical to the first but has a glucagon cartridge in place of theinsulin cartridge, is employed. The device is independently cannulizedand independently controlled for continuous subcutaneous infusion ofglucagon when desired.

In one embodiment, the patient is instructed to practice as follows.After taking a meal, the patient administers his prandial insulin (3-8units) and at the same time programs his glucagon pump to administerabout 162 μg of glucagon per hour subcutaneously over three hours andtimed to begin 2-3 hours after administration of his prandial insulin.In one embodiment, the above instructions are included in a kit with acomposition for the control of hypoglycemia. In this example, unlike inExample 1.A.ii, the continuous release of glucagon produces a smootherprofile with less of a peaked appearance and decay period than with asingle subcutaneous injection of glucagon. The increased availability ofglucagon during this patient's period of greatest susceptibility tohypoglycemia substantially decreases both the likelihood and severity ofsuch events. To offset the glucose-raising potential of subcutaneousadministered glucagon, the dosage of infused insulin can be increased tomaintain euglycemia during the period of glucagon administration. Byadministering glucagon in this way, the patient is provided withsufficient glucagon to serve as a cushion or buffer to the unopposedaction of insulin so as to prevent the risk of a hypoglycemic episode.Thus, the administration of glucagon enables the patient to maintaingood glycemic control without the excessive risk of frequent and severehypoglycemia.

B. Insulin Administered Parenterally

In one embodiment, glucagon can be administered by pump and insulinadministered parenterally, including by pump or other subcutaneousadministration. In one embodiment, pumps suitable for insulinadministration are also suitable for glucagon administration. Insulincan be administered parenterally as described in Example 1.A.i. Insteadof injecting glucagon as described in Example 1.A.i, however, the pumpis programmed (or actuated) to deliver glucagon continuously betweenhours 2 and 5 after the meal. The total dose of glucagon released isapproximately 121-324 milliunits over those three hours, this beingsufficient to provide protection from hypoglycemia. Alternatively, theamount of glucagon released is approximately 72-216 milliunits overthose three hours, this being sufficient to provide protection fromhypoglycemia.

C. Insulin Administered Transdermally [Including Patch and TopicalCream]

In one embodiment, insulin can be administered transdermally. Inaccordance with Example 1.C, the patient administers his insulin needsby use of transdermal patch (or cream). Instead of administering theglucagon parenterally as described in that example, however, the patientuses an insulin pump (containing not insulin but glucagon) to administerglucagon to prevent hypoglycemia in the early morning. Before going tobed, the patient programs his pump to deliver 50-120 milliunits ofglucagon to be administered subcutaneously between the hours of 01:00and 02:00, the period during which he is most susceptible tohypoglycemia. By so doing, the patient is able to maintain euglycemiausing the methods of the present embodiment, without the risk ofhypoglycemia occurring during his sleep.

D. Insulin Administered by Inhalation [Including Pulmonary, Buccal Nasaland Sublingual]

In accordance with Example 1.A.i, the patient administers insulin byinhalation rather than by subcutaneous injection. It will be understoodby those skilled in the art that similar methods can be employed wheninsulin is administered buccally, nasally or sublingually. The patientwill either continue to administer his basal need via GLARGINE (LANTUS),or he will utilize an insulin inhaler to administer basal insulin needs.The patient will administer his prandial insulin need (equivalent tobetween 5 and 10 units administered by subcutaneous administration)using his insulin inhaler (either pulmonary, nasally, buccally orsublingually). Instead of injecting glucagon as described in Example1.A.i, however, an insulin pump (containing glucagon rather thaninsulin) is programmed (or actuated) to subcutaneously deliver glucagoncontinuously between hours 2 and 5 after the meal. The total dose ofglucagon released is approximately 121-500 milliunits over those threehours, this being sufficient to provide the patient with protection fromhypoglycemia during his period of greatest susceptibility.Alternatively, the amount of glucagon released is approximately 72-216milliunits over three hours.

EXAMPLE 3 Co-Administration of Glucagon and Insulin, Admixed, by Pumpfor the Control of Diabetes and Prevention of Hypoglycemia

Some embodiments of the methods of the invention can also be practicedusing pump-based administration of an admixture of both insulin andglucagon. This method provides protection from hypoglycemia in directproportion to the amount insulin used and with a built in delay. It alsoreplaces basal levels of glucagon throughout the day and especiallyafter meals, as it will also be administered with the basal insulinadministered by the pump. This embodiment can be practiced usingstandard pumps currently available and described in Example 2. Onedifference is that the insulin cartridges used will contain a mixture ofinsulin and glucagon (optionally modified release glucagon), with theglucagon component being between 32-96 or 41-96 mU to be administeredeach hour of desired protection (prevention of hypoglycemia). In someembodiments, the amount of glucagon administered subcutaneously can beabout 0.1 ng/kg/min. of desired protection to about 20 ng/kg/min., 6ng/kg/min. of desired protection to about 20 ng/kg/min. of desiredprotection, and is in some embodiments 12 ng/kg/min.

The insulin/glucagon mixture is then administered by pump (for bothbasal and prandial insulin). The resulting glucagon (modified glucagon)plasma concentrations will map onto the insulin profile but with theattenuating characteristics of the glucagon variant used. This isillustrated in FIG. 7. This method will provide protection fromhypoglycemia over the period of susceptibility as required. In oneembodiment, the pump is equipped with a glucose sensor (see U.S. Pat.No. 5,474,552).

EXAMPLE 4 Co-Administration of Glucagon and Insulin, Orally, for theControl of Diabetes and Prevention of Hypoglycemia

The delivery of large molecules (e.g. proteins) orally is well known inthe art. Typically this involves enteric administration (see U.S. Pat.No. 5,641,515). In one embodiment, similar methods are used to deliverglucagon orally. In a typical scenario involving the oral delivery of amixture of insulin and glucagon, the patient takes an enteric tabletcontaining insulin appropriate to his prandial up to an hour beforeeating. The insulin component is designed for rapid onset once it beginsrelease. The glucagon component is designed to release later than theinsulin component, in one embodiment by 2-3 hours. Optionally, modifiedglucagon with a long half life can be used to ensure that glucagonlevels are elevated over an extended period. Administered in this way,the glucagon will be correctly and appropriately timed to preventhypoglycemia. In another embodiment, the patient administers his insulinusing any of the methods described herein and administers a glucagonpill as required, for example, with each of his meals, to preventhypoglycemia.

EXAMPLE 5 Compositions for Glucagon Suspension and Methods ofCharacterizing them

This Example demonstrates one composition of one embodiment of theinvention that was found to be useful for the storage of a glucagon anda method of one embodiment of the invention for verifying that thecomposition for storing glucagon provides the desired degree ofstability for the glucagon. GlucaGen® was mixed in 5% mannitol in WaterFor Injection at 200 μg/mL and 500 μg/mL in infusion pump cartridges.This solution was kept at 30° C. for set durations of time. Followingthe set durations, the pH and remaining percent glucagon were tested.The percent of remaining glucagon was determined by HPLC analysis. Theresults are presented below in Table 2 for 200 μg/mL GlucaGen® and Table3 for 500 μg/mL GlucaGen®. Results are from 3 sets of tests and the datais expressed as mean ±s.e.m.

TABLE 2 Remaining Glucagon (%) Time Point pH Relative to Time Zero TimeZero 3.36 ± 0.01 100.0 ± 2.3   3-hr 3.45 ± 0.04 101.1 ± 1.5   6-hr 3.53± 0.03 95.2 ± 4.3  9-hr 3.64 ± 0.04 99.1 ± 5.3 24-hr 3.79 ± 0.04 89.3 ±0.7

TABLE 3 Remaining Glucagon (%) Time Point pH Relative to Time Zero TimeZero 2.98 ± 0.00 100.0 ± 1.1  3-hr 3.10 ± 0.02 100.2 ± 1.6  6-hr 3.08 ±0.01  99.4 ± 0.9  9-hr 3.13 ± 0.03 101.2 ± 0.1 24-hr 3.21 ± 0.01  93.8 ±2.1

All solutions at the time of preparation were clear and colorless. Thesolutions remained clear and colorless throughout the compatibilitystudy. No visible particles were observed. Substantial amounts ofglucagon remain in the mannitol solution, even at high concentrationsand for all of the time periods examined, including 24 hours.Additionally, the higher concentration of glucagon resulted in a higherrelative percent of glucagon remaining. Thus, it appears that thiscombination is sufficient for maintaining glucagon in solution foruseful periods of time.

EXAMPLE 6 Compositions for Glucagon Suspension and Methods ofCharacterizing them

This example demonstrates a composition and one method of one embodimentof the invention by which such a composition can be analyzed todetermine the lifetime of a glucagon solution experiencing active, or“wear,” use. Samples of Glucagon Infusate at 200 μg/nL and 500 μg/mL in5% mannitol solution were prepared. An aliquot of approximately 3 mL ofeach Glucagon Infusate sample was added to an individual cartridge intriplicate. Cartridges were inverted 10 times and a 0.5-mL aliquot wasremoved from each as “Time Zero” for HPLC analysis. The filledcartridges were placed on a Platform Shaker set at 50 RPM and incubatedat 30° C. for 24 hours. At 3, 6, 9, 12 and 24 hours the cartridges wereremoved from the oven, inverted 10 times and a 0.5-mL aliquot wasdispensed from each for HPLC analysis. Appearance and pH were alsorecorded at each time-point.

All solutions at the time of preparation were clear and colorless. Thesolutions remained clear and colorless throughout the stability study.No visible particles were observed. Additional results are presented inTables 4 and 5.

TABLE 4 Remaining Glucagon (%) Time Point pH Relative to Time Zero TimeZero 3.47 ± 0.05 100.0 ± 2.7   3-hr 3.45 ± 0.03 85.7 ± 4.2  6-hr 3.47 ±0.03 83.6 ± 2.6  9-hr 3.50 ± 0.00 82.7 ± 2.9 12-hr 3.50 ± 0.03 75.5 ±6.0 24-hr 3.53 ± 0.01 48.9 ± 1.0

TABLE 5 Remaining Glucagon (%) Time Point pH Relative to Time Zero TimeZero 3.05 ± 0.02 100.0 ± 0.5   3-hr 3.01 ± 0.01 98.5 ± 1.2  6-hr 3.02 ±0.00 97.6 ± 0.7  9-hr 3.05 ± 0.00 95.1 ± 2.0 12-hr 3.03 ± 0.00 95.0 ±3.6 24-hr 3.00 ± 0.00 93.1 ± 1.1

With continuous shaking at 50 RPM, GlucaGen® in 5% mannitol at 200 μg/nLshowed 25% glucagon loss after being stored at 30° C. for 12 hours, andabout 50% glucagon loss after being stored at 30° C. for 24 hour.GlucaGen® in 5% mannitol at 500 μg/nL exhibited 7% loss over 24 hours.Thus, even with lower concentrations of glucagon, e.g. 200 μg/nL, asubstantial amount of glucagon remains in solution after extendedperiods of time. Furthermore, it appears that the greater concentrationof glucagon results in a more stable formulation, even through activeshaking of the formulation for 24 hours.

In one embodiment, the glucagon or variant thereof, is stored at highconcentrations. High concentrations may be, for example, greater than100 micrograms/mL. In one embodiment, high concentrations are more than200, 200-300, 300-400, 400-500, 500, 500-600, 600-800, 800 or more μg/nLglucagon, up to the saturation (or super-saturation) limit. In oneembodiment, less active forms of glucagon are stored at higherconcentrations. This allows for greater stability of the glucagonsolution and for one to administer a larger volume of glucagon solutionto a recipient.

EXAMPLE 7 Very Low Dose (VLD) Glucagon can be Used to Elevate BloodGlucose in Insulin-Treated Diabetic Patients

This example demonstrates the effectiveness of applying a low dose ofglucagon to a patient via a particular route of administration toelevate the blood glucose level of the patient. This example describesclinical testing in which the level of glucagon required to increaseglucagon and glucose blood levels was examined. As explained in moredetail below, the results show that the methods of some embodiments ofthe invention can be used to avert insulin induced hypoglycemia.

A. 0.8 to 4 ng/kg/min. of Glucagon

Six patients with Type 1 diabetes were stabilized overnight at bloodglucose levels of 90 to 120 mg/dl in a nocturnal titration period.Stabilization of blood glucose levels in this range of 90 to 120 mg/dLwas accomplished using insulin doses that generally varied between 0.65to 1.4 units/hour.

The following morning, these patients were administered glucagonsubcutaneously by continuous infusion pump over a range of 0.8 to 4.0ng/kg/min. (specific doses were 0.8, 1.6, 2.4, 3.2 and 4.0 ng/kg/min) ofglucagon along with their usual doses of insulin. Each dose level withinthis range was infused for 2 to 3 hours. Under this lower range ofdoses, 0.8-4.0 ng/kg/min. of glucagon, there were no explicit consistenttrends in the glucagon or glucose levels of the patients.

B. 8 to 16 ng/kg/min. of Glucagon

After undergoing a nocturnal titration period in which their bloodglucose levels were maintained between 90 and 120 mg/dl, six patientswere dosed with glucagon in a range of 8 to 16 ng/kg/min. Glucagon wasadministered subcutaneously by continuous infusion pump. The specificdoses administered were 8, 12 and 16 ng/kg/min; each dose level wasinfused for 3 hours. Patients were maintained on their standard basallevels of insulin by continuous subcutaneous pump administration priorto the administration of the glucagon.

The 8 ng/kg/min. dose of glucagon did result in a significant rise inboth glucose and glucagon, approximately doubling the blood glucagonlevels and resulting in an increase of approximately 40% for bloodglucose levels. Thus, a dose of 8 ng/kg/min. can elevate blood glucoselevels in these patients. Moreover, even those lower ranges tested,while not sufficient for elevating blood glucose levels under these testconditions, could be sufficient for preventing hypoglycemia inducedthrough insulin administration in some patients.

In 3 patients, an infusion of 12 ng/kg/min. of glucagon subcutaneouslyfor 9 hours resulted in elevated plasma glucagon levels that weresustained for 6 to 8 hours in the range of 100 to 200 pg/ml (see FIG.8). The peak mean plasma level at the 12 ng/kg/min. dose was 185 pg/ml.The blood glucose level of the patients was also substantially elevated(see FIG. 9). This clearly demonstrates that sustained, very low, dosesof glucagon can be administered to patients, effectively, over aprolonged period of time with the desired result of controllablyelevating blood glucose levels.

Two patients were treated with 16 ng/kg/min. of glucagon subcutaneouslyfor 9 hours. In both subjects, dosing with 16 ng/kg/min. resulted inhigher peak plasma glucagon levels than were achieved on the 12ng/kg/min. dose (see FIG. 8). In both subjects, the peak level wasachieved during the first 3 hours of the infusion and then tended todecrease to levels seen at with the 12 ng/kg/min. dose. At the 16ng/kg/min. dose level the peak mean plasma level was about 254 pg/mL.Despite the decline in glucagon levels during the latter part of theinfusion, glucose levels remained elevated throughout the course of theinfusion (see FIGS. 8-11). Glucose levels achieved at the 16 ng/kg/min.dose were generally higher than those achieved at the lower dose of 12ng/kg/min. (see FIG. 9).

Subjects described above were generally maintained on their basal rateof insulin throughout the fasting evaluation period. The insulinadministered varied between patients and over time from 0.5 to 1.8 unitsof insulin per hour. Glucose levels began to rise as early as 30 minutesafter the glucagon infusion was started. On average, this rise wassustained throughout the glucagon infusion.

These results demonstrate that very low doses of continuouslyadministered glucagon can be used to increase blood glucose levels ininsulin treated patients not experiencing hypoglycemia.

These data confirmed that the administration of a dose of glucagon inthe range of 8 to 16 ng/kg/min. yields an increase in the plasmaglucagon levels in the range of about 100 to 200+ pg/mL and that theselevels can be maintained over a substantial period of time. The normalreference range for glucagon is 50 to 150 pg/mL. As will be appreciatedby one of skill in the art, these results demonstrate that low,continuous dosing of glucagon can elevate blood glucose levels indiabetic patients and so provides a means to prevent hypoglycemia inthose patients. These results demonstrate the amount of glucagonrequired to induce elevated blood glucose levels. One of skill in theart will appreciate that lower glucagon doses may be used to preventhypoglycemia in other patients, but the doses of 8 ng/kg/min. and higherexemplified here are effective in preventing hypoglycemia. The actualminimal amount can vary between patients, and given the presentdisclosure, one of skill in the art can readily determine what theminimal amount can be for a particular patient.

Interestingly, the levels achieved in the present example with VLDglucagon in amounts of 12 to 16 ng/kg/min are similar to the levels seenin non-diabetics in response to conditions of experimentally-inducedhypoglycemia.

The following Example demonstrates how these doses of glucagon canprevent hypoglycemia even when there is an insulin challenge, i.e., adose of insulin that would otherwise induce hypoglycemia in the patient.

EXAMPLE 8 Prevention of Insulin Induced Hypoglycemia with Glucagon

This example demonstrates that insulin induced hypoglycemia (a bloodglucose less than 50 mg/dl for this example) can be prevented with lowdoses of continuously administered glucagon. The study was arranged inthree visits. The first visit involved increasing the amount of insulinadministered to the subjects without supplying any glucagon; thesubjects' blood glucose levels were monitored. This first study definedthe insulin challenge needed to induce hypoglycemia in these patients.In the following two visits, two different doses of glucagon wereadministered to the patients and the patients' blood glucose levelsmonitored to determine whether the glucagon prevented or delayed anyhypoglycemia that would have otherwise been induced by the insulinchallenge. Thus, by comparing the glucose levels measured in the firstvisit with those in the later two visits, one is able to determinewhether the glucagon prevented hypoglycemia.

During the first visit, each subject's basal insulin rate was titratedupward (in 50% increments every 90 minutes (for example, from a basalrate of 1.0 unit/hour, to 1.5 units per hour for 90 minutes, to 2.0 for90 minutes, to 2.5)) to induce hypoglycemia (blood glucose less than 50gm/dl). The results for one patient are shown in FIG. 12, with visit 1represented by squares. The lower set of lines trace the increases ordecrease of insulin administered to the subject. The insulinadministered was returned to basal rate of infusion (and glucoseadministered) after 12:30; thus, the spike after 12:30 is to beexpected. Blood sugar levels were allowed to vary between 50 mg/dL and350 mg/dL. Glucose levels lower than 50 mg/dL were considered indicativeof hypoglycemia.

During visits 2, and 3, subjects received a continuous infusion ofvarious doses of glucagon (starting at 7:00 a.m. and ending around 4:00p.m.) to determine whether that dose would avert the insulin-inducedhypoglycemia or delay the time to onset of hypoglycemia that wasobserved in the first visit. Because the onset of action on glucoselevels is around 60 to 90 minutes, the glucagon infusion was started onehour before the insulin infusion rate was stepped up.

Two different doses of glucagon were examined, a dose at 8 ng/kg/min.(triangles) and a dose at 16 ng/kg/min. (circles). The results of theselow doses of continuous glucagon on blood glucose levels are shown inFIG. 12. As shown in FIG. 12, the 8 ng/kg/min. dose of glucagon(triangles) maintained a higher level of blood glucose in the patientcompared to the test in which no glucagon was administered. Again, thecontrol line, (no glucagon, only increasing insulin, squares)effectively ended at 12:30; past this point on the graph, no insulin wasadministered, and thus the comparison should be made between 7:00 and12:30. Hypoglycemia was delayed for approximately two hours.

As shown in FIG. 12, the 16 ng/kg/min. dose (circles) maintained bloodglucose levels substantially above the control level and even above the8 ng/kg/min. (triangles) glucagon dose for a substantial period of time.Additionally, while the 8 ng/kg/min. dose was able to preventhypoglycemia and kept blood glucose levels above 50 mg/dL, the 16ng/kg/min. dose maintained blood glucose levels closer to 100 mg/dL.This indicates the effectiveness of both of these ranges, as well asdose dependency.

The lines in the lower section of the graph (i.e., triangle, square, andcircle marked lines beneath the 50 mg/dl line in FIG. 12) represent thesteps in insulin infusion over the time of the test. Thus, the lowersquare marked line is indicative of insulin levels during visit 1, andthe lower circle and triangle marked lines are indicative of insulinlevels during visits 3 and 2 respectively.

In another patient, the effect of the 8 ng/kg/min. dose was not aspronounced as that shown in FIG. 12. Thus, this patient would require ahigher dose or infusion rate to prevent hypoglycemia. This isdemonstrated by the 16 ng/kg/min. dose data, which demonstrated aneffect similar to that shown in FIG. 12 for the first patient, i.e.,elevated blood glucose levels above 200 mg/dl.

In addition to the above glucagon levels tested, the effectiveness of alower level of glucagon was also tested, using a similar protocol. Inanother patient examined, the above experiment was repeated with a lowerdose of glucagon, 4.0 ng/kg/min., and a slightly lower upper limit ofinsulin (1.5 fold the basal rate of insulin). The results are displayedin FIG. 13. As can be seen in the graph, while an increase in the BRIduring a first visit (diamonds) resulted in lower blood glucose levels(squares), the administration of 4 ng/kg/min. of glucagon in a secondvisit (asterisk) helped to maintain elevated blood glucose levels (“X”)as well as delay the decrease in blood glucose levels, even when insulinlevels were increased (circles). In this patient, while the raised levelof insulin would have otherwise induced hypoglycemia, the presence of4.0 ng/kg/min. of glucagon was sufficient to prevent hypoglycemia fromoccurring.

In addition to the above identified benefits of administering low dosesof glucagon to prevent hypoglycemia, there appears to be a prolongedbenefit to this dosing schedule for subcutaneously administeredglucagon. Blood glucose levels remained elevated even after the glucagonhad ceased to be administered (see the section of FIG. 12 following 4:00p.m.). Thus, continuous administration of glucagon is not required tomaintain an extended period of elevated blood glucose levels.

The above methodology can be used to determine the appropriate amount ofglucagon to administer to a particular patient. For example, testingpatients who take greater amounts of insulin, for example 2-10, 10-20,20-30, or more units of insulin per hour in this fashion can be used todetermine the appropriate dosing regimen for glucagon to preventhypoglycemia. In the above example, insulin amounts were increased by1.5 to about 2.5 fold. In one embodiment, one increases the amount ofglucagon proportionally as one increases the amount of insulin and thendetermines (via the above methodology) if the new glucagon dose iscorrect to prevent hypoglycemia. In this fashion, one can determine theappropriate dosage of glucagon for a particular patient. One of skill inthe art will recognize that this relationship need not be 100%proportional and that some degree of routine experimentation can beuseful in optimizing the final values. Additionally, one can use theabove example to determine a dosing schedule to see how frequentlyglucagon actually needs to be administered. In some embodiments, ratherthan constant administration, glucagon is administered only 99-90,90-80, 80-60, 60-40, 40-20, 20-10, 10-1 percent, or less of the time.

As will be appreciated by one of skill in the art, variations in themethod of administration (e.g. patch, inhalation, topical creams, I.V.,etc.) can also be examined using this methodology. Additionally, as willbe appreciated by one of skill in the art, this methodology can bemodified to determine the appropriate timing of the administration ofglucagon to the patient. For example, the glucagon can be administered 1hour before the insulin level is adjusted, and can be increased to 90minutes or more or decreased to 45 minutes or less, for example, todetermine if better control of glucose blood levels is maintained. Aswill be appreciated by one of skill in the art, the time ofadministration and method of administration can influence the doserequirements. Additionally, the importance of various additives can alsobe examined through the above methodology.

EXAMPLE 9 Determining an Amount of VLD Glucagon for the Prevention ofHypoglycemia

This example describes a clinical trial for demonstrating that glucagonadministered in accordance with the present methods will function asdesired in maintaining a desired blood glucose level. The method alsoprovides means to determine the optimal ratio of insulin to glucagon foradministration to a particular patient to prevent insulin-inducedhypoglycemia.

Subjects report in the evening and are fed a standard meal atapproximately 1700 to 1800 hr. They are instructed to bolus their usualdose of insulin through their pump based on carbohydrate counting. At2000 hr, another pump (Pump 2) is initiated on the contra-lateral sideof the abdomen with saline infused at the same rate as the insulininfusion. From approximately 2200 hr, subjects fast and are on theirusual basal dose of insulin through CSII. Plasma glucose is checked witha YSI throughout the night every hour (or more frequently if necessary).The plasma glucose is maintained between approximately 100 and 125 mg/dlthrough adjusting the basal insulin rate and, if necessary, with the useof IV 5% Dextrose (if the plasma glucose decreases below 90 mg/dl) andIV Insulin (if their plasma glucose increases above 160 mg/dl).

From 0700 to 0800 hr, baseline blood samples are drawn and tested forplasma glucose, free fatty acids, ketone bodies, glucagon and insulinlevels. At 0800 hr, subjects receive two times their usual basal dose ofinsulin through their Insulin Pump 1 to induce controlled hypoglycemia.Through Animas Pump 2, they receive VLD glucagon on the opposite side ofthe abdomen. The dose of glucagon administered is individualized basedon previous studies. The selected dose is the highest dose that does notcause hyperglycemia (>180 mg/dl) in each individual study subject.

From 0800 to 1200 hr. blood is drawn every 5 to 10 minutes and YSIplasma glucose is determined. Blood is also drawn to measure free fattyacids, ketone bodies, glucagon and insulin levels every 15-30 minutesfrom 0800 to 1200 hr. Despite receiving a much higher than basal dose ofinsulin through CSII (continuous subcutaneous insulin infusion), theplasma glucose levels of the study subjects does not decrease tohypoglycemic levels (<60 mg/dl), due to the simultaneous continuoussubcutaneous infusion of glucagon, which counteracts the glucoselowering effects of the high basal dose of insulin. If during the study,it is found that the subject's plasma glucose begins to decrease andconsistently falls below 90 mg/dl on 2 consecutive occasions, the doseof the continuous glucagon infusion is titrated upwards by 25% tocounter the fall in plasma glucose. On the other hand, if the subject'splasma glucose begins to increase and rises more than 25% in 1 hour, thedose of the glucagon infusion is titrated downwards by 25% to counterthe increase in blood glucose. Thus, this trial demonstrates that thecombination of a small amount of glucagon with insulin can preventinsulin-induced hypoglycemia. Both the insulin and the glucagon can beadministered subcutaneously.

By the above methodology, one can also test various forms of glucagonformulations and methods of administration. Thus, one can determine theoptimal timing and mode of delivery for glucagon or a glucagon mimeticor variant, or formulations of them. One can also use these methods totest the suitability of other hypoglycemic and hyperglycemic substancesfor the methods and compositions herein disclosed.

EXAMPLE 10 Preventing Nocturnal Hypoglycemia

This example demonstrates one method for demonstrating that acomposition, for example, of glucagon, glucagon variants, orformulations thereof, is effective in preventing blunt insulin inducednocturnal hypoglycemia in humans. Additionally, it provides a method fortesting the effectiveness of a glucagon or variant or formulation ofeither for preventing nocturnal hypoglycemia.

Two hours (˜1800 h) after a standardized dinner meal, subjects receivetwice their normal dose of insulin to induce the development ofnocturnal hypoglycemia via a first pump. At 2200 h, they receive aninfusion of the glucagon via a second pump (CSI). The blood glucoselevel is then monitored throughout the night. An adequate dose ofglucagon prevents the blood glucose levels from decreasing tohypoglycemic levels. Both the glucagon and insulin can be administeredsubcutaneously.

EXAMPLE 11 Administering Low Doses of Glucagon to Prevent Loss ofHypoglycemic Awareness

To prevent loss of hypoglycemic awareness in a subject taking insulin,one administers to that subject more than 5 to about 16 ng/kg/min ofglucagon subcutaneously to the subject to prevent the blood glucoselevel from entering an undesirable, hypoglycemic level (e.g. less than50 mg/dL). To determine that the dose administered is sufficient, bloodglucose levels can be measured throughout various time points in a testperiod to ensure that the subject's glucose level does not drop below acertain point (e.g., less than 50 mg/dL). Such testing can also be usedto optimize the dose of glucagon administered to the patient. Theadministration of glucagon is chronic, i.e., through the preventiveadministration of a low dose of glucagon, a patient will avoidhypoglycemia and so not develop hypoglycemic unawareness due to repeatedhypoglycemic episodes through prolonged insulin use. Alternatively, onecan administer between 0.1 to about 20 ng/kg/min or 4.0 to about 16ng/kg/min. of glucagon to the patient.

EXAMPLE 12 Administering Low Doses of Glucagon to Recover HypoglycemicAwareness

A patient suffering from a loss of hypoglycemic awareness can beidentified by screening to determine if the patient can identify whenhis or her blood glucose levels have dropped below 70 mg/dl of bloodglucose. Once identified, one then administers an appropriate dose, inone embodiment that dose is 8-16 ng/kg/min., of glucagon subcutaneouslyto the subject to prevent the blood glucose level from declining to alevel (e.g. less than 50 mg/dL).

Blood glucose levels can be taken throughout various time points todetermine that the subject's glucose level has not dropped to ahypoglycemic point. These measurements can also be used to optimize theamount of glucagon administered to the patient. Through theadministration of glucagon, a patient will experience fewer hypoglycemicepisodes, and his or her awareness of hypoglycemia will improve. Thesubject's awareness of hypoglycemia can be tested, as described above,by determining if the subject can identify when his or her blood glucoselevel is below a certain point (e.g., 70 mg/dl).

Various doses, various forms of formulations, and various methods ofapplication (administration) can all be tested using the abovemethodology to determine the optimal dosage, or if a formulation ormethod of administration is optimal for the prevention of hypoglycemia.

EXAMPLE 13 Co-Administration of Glucagon Transdermally and Insulin forThe Control of Diabetes and Prevention of Hypoglycemia [Including Patchand Topical Cream]

The use of transdermal patches for the delivery of therapeutic drugs isincreasingly more common. Patches provide a non-invasive and easy methodof delivering some drugs to the bloodstream. Nicotine and hormonereplacement therapies are perhaps the best known uses of thistechnology. One of the characteristics of drug delivery by transdermalpatch is that the rate of delivery is typically constant and persistsfor a long period of time (as long as the patch is worn). Thischaracteristic has proven beneficial in the area of pain management(FENTANYL) and nicotine replacement therapy, in which long duration flatprofiles are ideal. This characteristic makes the transdermal patchsuitable for basal replacement of insulin or glucagon. See PCT patentpublication No. WO0243566, incorporated herein by reference.

Fast-acting patches are also known. The delivery of proteins (insulin inparticular) transdermally into the bloodstream in well under an hour isreported in U.S. Patent No. 5,707,641, incorporated herein by reference.The ability to deliver other proteins in the same way and using similarformulations is also recited. Glucagon can accordingly be administeredin such a manner.

Development of insulin patches is currently being pursued by HelixBioPharma, from Canada, and IDEA in Germany, where phase II trials arecurrently in progress. The IDEA technology (TRANSFEROMEs®) is directedto the transport of large molecules, such as peptides, across the dermalbarrier. FIG. 6 illustrates the effect of molecular weight andlipophilicity on the rate of transdermal transport in case of permeation(upper and lower gray curve for the more or less lipophilic substances,respectively) or of the TRANSFEROME® mediated penetration (black lineand bullets). Gray bullets represent the commercial drugs in transdermalpatches. Regardless of the technology, the ability to efficientlytransport peptides transdermally is proven and imminent. In particular,both insulin and glucagon can be delivered transdermally, thus providingsome embodiments of the present invention that are practiced usingtransdermal patches and similar devices.

A variety of possible patch structures and matrices can be employed,with the specific type selected according to the specific mode of useintended. For example, one can employ two basic types of insulin matrix,one for basal insulin replacement and one for prandial insulinreplacement. Glucagon patches can be formulated to provide post-prandialglucagon for protection from hypoglycemia or basal glucagon replacement.In one embodiment, the invention can be practiced using patch matricescomprising combinations of these basic types, either together in thesame matrix or separately in sub-matrices.

Thus, the following patch matrices can be useful in the practice of someembodiments:

-   -   a matrix containing insulin for basal insulin replacement;    -   a matrix containing insulin for prandial insulin replacement;    -   a matrix containing glucagon for basal glucagon replacement;    -   a matrix containing glucagon for post-prandial protection from        hypoglycemia;    -   a matrix containing insulin for prandial insulin replacement and        glucagon for post-prandial protection from hypoglycemia;    -   a matrix containing insulin and glucagon for basal replacement        of both insulin and glucagon; and    -   matrices which are composed of 2 or more sub-matrices, each sub        matrix being one of the matrices described above.

Topical creams can be used as an alternative to a patch.

For prandial insulin matrices, short acting insulins such as LISPRO(HUMALOG), ASPART (NOVOLOG), or GLULISINE (APIDRA) can be used. Theprandial insulin matrix is typically applied at or at some time beforemealtimes, according to its rapidity of onset. A prandial insulin patchwhich minimizes the time to onset can be used so that the patch isapplied near to mealtimes. The time to onset depends on the insulinconcentration and the nature of the formulation. For example, a simplewet-matrix of insulin has slower onset than an insulin patch formulatedaccording to U.S. Pat. No. 5,707,641. Monomeric insulin will act fasterand be more easily absorbed than larger clusters of insulin molecules,because molecular size impacts bioavailability from transdermal patches.

A number of different methods of managing the delivery of prandialinsulin can be utilized. For example, patches of differentconcentrations can be used for fixed periods of time, and theconcentration selected by the patient would depend on the amount ofcarbohydrate eaten. The duration for which the patch is worn could befixed. The patch would not necessarily be exhausted on removal, i.e. itcould deliver a fixed concentration throughout its use. As anotherexample, single concentration prandial patches could be employed asfollows. The time the patch is worn is varied according to the amount ofcarbohydrate eaten. The patch would not necessarily be exhausted onremoval, i.e. it could deliver a fixed concentration throughout its use.

As another example, prandial patches containing fixed doses of insulincould be used. The advantage of a self-exhausting patch is that failureto remove them does not of itself carry the risk of hypoglycemia. Such apatch would be substantially exhausted on removal and the rate ofinfusion would be front loaded. In one embodiment, the prandial insulinpatch used has a variable insulin concentration (appropriate to theamount of carbohydrate consumed), has a very rapid onset (preferablyimmediate but not more than one hour), is removed or deactivated after afixed length of time (preferably from between 3 and 5 hours) andactivated at (or no longer than one hour before) mealtimes. Prandialglucagon patches are applied at mealtimes or at some predetermined timeafter the meal according to the rapidity of onset associated with thepatch. The rate of onset is determined both by the concentration of theglucagon used and the nature of the formulation used. For example, asimple wet-matrix of glucagon would be expected to have a slower onsetthan a glucagon patch formulated according to the techniques describedin U.S. Pat. No. 5,707,641.

In one embodiment, the glucagon patch is constructed so that peak outputof glucagon is reached at some time between 2 and 5 hours afterapplication. This patch is applied at mealtimes. The amount of glucagonin the patch will be an amount sufficient to deliver to the patient anamount equivalent to more than 5-30 ng/kg/min., and even morepreferably, 8-16 ng/kg/min. of glucagon administered subcutaneously. Insome embodiments, the amount of glucagon administered is between 0.1-30ng/kg/min.

A number of different patch constructions can be used. These include:

-   -   a patch containing a single matrix or set of sub-matrices in a        single compartment;    -   a patch containing 2 or more separate compartments each        containing its own matrix or set of sub-matrices, the patch        being activated or deactivated as a single unit; and    -   a patch containing 2 more separate and independent compartments,        each containing its own matrix or set of sub-matrices, in which        each compartment is independently activated and deactivated.

Other patch configurations can be employed, and practice of theinvention is not limited to the configurations described above.

A. Insulin Administered Transdermally [Including Patch and TopicalCream]

(i) Insulin Administered Transdermally [Including Patch and TopicalCream] and by Subcutaneous Injection

In this example only prandial insulin and prandial glucagon areadministered by transdermal patch. This can be achieved in a variety ofways, including: (i) use of a single matrix of glucagon and insulinadmixed; (ii) use of a single compartment with two sub matrices, onecontaining insulin and the other containing glucagon; (iii) use of asingle patch containing two compartments, one containing insulin and theother containing glucagon, both compartments being activated anddeactivated simultaneously; and (iv) use of two separate patches (or twocompartments in a unitary patch), one containing insulin and the othercontaining glucagon, each patch or compartment being independentlyactivated and deactivated. Basal insulin is delivered parenterally asdescribed in Example 1.A.i by subcutaneous injection of a long-actinginsulin such as GLARGINE or ULTRALENTE. In one embodiment, method (i) isused. If different matrices are used to achieve the desiredpharmacokinetics, then method (ii) or (iii) can be used. If the timingof insulin onset and glucagon onset is not matched, then method (iv) canbe used.

In this illustrative example, method (ii) above is used. The useractivates the prandial patch at mealtimes (or preferably within one hourbefore mealtimes), thereby activating both sub-matrices at the sametime. If a fixed concentration patch is used, the user removes the patchafter a period of time proportionate to the amount of carbohydrateingested. If a variable concentration patch is used, then the userremoves the patch after a fixed period of time, typically between 1 and3 hours after eating. In one embodiment, fixed concentrations are used.In such an embodiment, the amount of glucagon administered can beincreased with the amount of insulin administered.

(ii) Insulin Administered Transdermally [Including Patch and TopicalCream]

In this example, both the insulin and glucagon are administeredtransdermally. Two different types of patch (or independently actuatedcompartments) can be employed. One patch (or compartment) contains amatrix designed to replace basal insulin over a 24 hour period. A singlepatch (or compartment) containing both the prandial insulin and glucagonin separate sub-matrices with onset times appropriate to applying theprandial patch (or activating the prandial compartment) at (or near)mealtimes can be used.

In one embodiment a unitary device containing four independentlyactuable compartments is used, one containing basal insulin, which isactivated on application and left active for 24 hours, and the other 3compartments containing the prandial insulin and glucagon in separatesub-matrices within the same compartment, each compartment beingseparately activated at mealtimes and deactivated at some time after themeal, the time of activation being proportional to the amount ofcarbohydrate consumed.

On beginning a meal (or at some time up to an hour before the meal), thepatient activates one of these prandial compartments [e.g. by pullingaway a hermetic plastic seal between the patch and the skin], a processwhich initiates the transdermal infusion of the insulin and glucagon. Atsome later time, a period in direct proportion to the amount ofcarbohydrate taken, the prandial compartment is deactivated [e.g. byreplacing the barrier used to activate the compartment or the totalremoval of that compartment from the end of the patch]. The insulinformulation in the insulin sub-matrix is short acting insulin, and thepatch is designed for rapid onset. The glucagon formulation in glucagonsub-matrix is designed to reach efficacious concentrations in thebloodstream between 1 and 3 hours after activation of the compartment,hence providing protection from hypoglycemia at the appropriate part ofthe cycle as described in Example 13.A.i.

In an alternative embodiment, a unitary device which allows for morethan 3 meals a day may easily be devised by allowing for more than 3prandial compartments. In an alternative embodiment, as described above,the prandial drugs may be contained in totally separate (andindependently actuated) prandial patches. In an alternative embodiment,the prandial patch may consist of separate insulin and glucagoncompartments so that each may be independently activated anddeactivated.

A unitary device containing separate and independently controlledcompartments for insulin and glucagon could have 7 separatecompartments, one for basal insulin, 3 for prandial insulin, and 3 forprandial glucagon. The basal patch is designed to replace basal insulin(worn for 24 hours before being replaced). The insulin used may be anyinsulin suitable for transdermal delivery. The basal insulin compartmentmay also optionally contain an amount of glucagon (admixed or in asub-matrix) sufficient to supply basal glucagon over each 24 hourperiod. This would have the beneficial effect of providing protectionfrom hypoglycemia throughout the day and in particular during sleep.

The amount of glucagon in the patch can be an amount sufficient todeliver to the patient an amount equivalent to more than 5-30 ng/kg/min.of subcutaneously administered glucagon, for example, more than 6-20ng/kg/min., and for another example, 8-16 ng/kg/min. of glucagon. Inother embodiments, the amount administered is an amount equivalent to0.1 to 30 ng/kg/min or 4.0 to 20 ng/kg/min. administered subcutaneously.Greater amounts of glucagon can be present when more than 0-3 Units ofinsulin are to be administered. For example, a greater amount ofglucagon can be present when 3-20 Units of insulin are to be applied inone hour. Alternatively, the same amount of glucagon is presentregardless of the amount of insulin.

B. Insulin Administered by Inhalation [Including Pulmonary, Buccal Nasaland Sublingual]

In this example, insulin is delivered by inhalation, as described above.Inhalation can be the mode for delivery of only the prandial insulindelivery (basal being delivered parenterally), or all insulins used canbe delivered by inhalation. The patient administers an amount of insulinappropriate to his or her meal by inhalation (in one or moreactuations). The patient can optionally increase the insulin after ameal as appropriate.

The glucagon is administered by patch as described in Example 13.A.i.The patch (or set of glucagon compartments in a unitary patch) isattached to the skin, and the patch or (sub compartment) is activated atmealtimes. The patch is designed to have slow onset, so that theglucagon is only present in the body in efficacious quantity after 2hours. The patch is worn for 4 hours before being removed, the residualglucagon in the body being sufficient to provide protection fromhypoglycemia over the required period of 2-5 hours.

In one embodiment, as described herein, the glucagon in the patch is along acting glucagon (e.g. iodinated glucagon). The patch may then, insome embodiments, be worn for a shorter time while still ensuring thatthe protection afforded by the modified glucagon is provided over aperiod of 2-5 hours.

In another embodiment, the user can apply the glucagon by means of atransdermal cream, which acts similarly to a transdermal patch (anamount equivalent to at least about 8-16 ng/kg/min. of glucagonadministered subcutaneously can be administered through the cream). Theformulation of such a cream can differ from the formulation used in apatch but perform essentially the same function. When glucagon isadministered in this way, it may be advantageous to encapsulate theglucagon in liposomes or TRANSFEROMEs® to prevent the supply of glucagondrying on the skin and reducing bioavailability.

C. Insulin Administered Parenterally

In accordance with Example 1.A.i, the patient's insulin needs are met byparenteral administration. The glucagon is administered by patch asdescribed in Example 13.A.i. The patch (or set of glucagon compartmentsin a unitary patch) is attached to the skin and the patch or (subcompartment) is activated at mealtimes. The patch is designed to haveslow onset, so that the glucagon is only present in the body inefficacious quantity after 2 hours. The patch is worn for 4 hours beforebeing removed, the residual glucagon in the body being sufficient toprovide protection from hypoglycemia over the required period of 2-5hours.

In one embodiment, as described herein, the glucagon in the patch is along acting glucagon (e.g. iodinated glucagon). The patch may then beworn for a shorter time while still ensuring that the protectionafforded by the modified glucagon is provided over the required periodof 2-5 hours.

In an alternative embodiment, the user may apply the glucagon by meansof a transdermal cream, which acts similarly to a transdermal patch. Theformulation of such a cream can differ from the formulation used in apatch but performs essentially the same function. When glucagon isadministered in this way, it may be advantageous to encapsulate theglucagon in liposomes or TRANSFEROMEs® to prevent the glucagon fromdrying on the skin and reducing bioavailability.

D. Insulin Administered by Pump

In this example, the patient's insulin needs are administered by pump asdescribed in Example 2. The glucagon is administered by patch asdescribed in Example 13.A.i. The patch (or set of glucagon compartmentsin a unitary patch) is attached to the skin and the patch or (subcompartment) is activated at mealtimes. The patch is designed to haveslow onset, so that the glucagon is only present in the body inefficacious quantity after 2 hours. The patch is worn for 4 hours beforebeing removed, the residual glucagon in the body being sufficient toprovide protection from hypoglycemia over the required period of 2-5hours.

In one embodiment, the glucagon in the patch is a long acting glucagon(e.g. iodinated glucagon). The patch may then be worn for a shorter timewhile still ensuring that the protection afforded by the modifiedglucagon is provided over the required period of 2-5 hours.

In an alternative embodiment, the user may apply the glucagon by meansof a transdermal cream, which acts similarly to a transdermal patch. Theformulation of such a cream may differ from the formulation used in apatch but performs essentially the same function. When glucagon isadministered in this way, it may be advantageous to encapsulate theglucagon in liposomes or TRANSFEROMEs® to prevent the glucagon fromdrying on the skin and reducing bioavailability.

EXAMPLE 14 Co-Administration of Glucagon by Inhalation and Insulin forthe Control of Diabetes and Prevention of Hypoglycemia [IncludingPulmonary, Buccal, Nasal and Sublingual]

A number of dry powder inhalation technologies are currently indevelopment, including: Aradigm's AERx®, Inhale Therapeutics' Exubera®,Alkermes' and Eli Lilly's AIR, Insulin Technospheres (Mannkind/PDC), andAerogen's and Disetronic's Aerodose. Methods and devices for deliveringinsulin to the pulmonary alveoli, where it may be absorbed into theblood stream, are described in U.S. Pat. Nos. 5,997,848; 6,131,567;6,024,090; 5,970,973; 5,672,581; 5,660,166; 5,404,871; and 5,450,336.The main difficulties that had to be overcome to enable aerosolmacromolecular delivery were: low system efficiency (bioavailability);low drug mass per inhalation (c.f asthma); and poor dosingreproducibility.

One relevant factor is efficiency (bio-availability). Bioavailabilitydepends primarily on the aerosol particle size (most existing systemsonly deliver 10%-20% of the drug administered to the alveoli) ratherthan on the nature of the drug being administered. When the drug beingdelivered actually reaches the alveoli, its bioavailability is then veryhigh almost regardless of the drug in question. Because the technicalproblems (and solutions) associated with delivering insulin are similarto those for delivering glucagon, the solutions enabling delivery ofinsulin are directly applicable to similarly sized macromolecules likeglucagon. One embodiment provides dry powdered formulations prepared byadmixing insulin and glucagon. The use of inhalers for deliveringinsulin is primarily aimed at supplying rapid insulins for prandialpurposes. Long acting insulins can be delivered by inhalation ifdesired.

Some embodiments can be practiced using inhalers in a number of ways,including with insulin and glucagon in separate inhalers; with insulinand glucagon admixed in a fixed ratio in an inhaler; with a dual chamberinhaler in which insulin and glucagon are administered separately; andwith dual chamber inhalers in which insulin and glucagon areadministered simultaneously. Because prandial inhalers typically containrapid acting insulins, they are unsuitable (in the way that insulinpumps are) for the delivery of basal insulin. A separate pump or chambercan be provided if both prandial and basal insulins are to be deliveredby inhalation.

A. Insulin Administered by Inhalation [Including Pulmonary, Buccal Nasaland Sublingual]

The hypothetical patient administers basal insulin using ULTRALENTE bysubcutaneous injections at a dosage level of 20 units administered atbedtime. Alternatively, he may choose to administer the same drug (in adose that would provide a daily bioavailability of 20 units) byinhalation. It may also be beneficial or desirable for him to administerthe basal dose by inhaler at a number of times during the day, forexample, at mealtimes in addition to bedtime. Because there is a slightdelay (approximately 20 minutes) before insulin attains significantserum concentration when compared to subcutaneous delivery, the userwill administer his prandial insulin requirement approximately 20minutes before eating. He does this by administering between 25 and 50units (assuming a bioavailability of approximately 20%) of insulin bymeans of a metered dose inhaler.

The inhaler may be dose alterable (see U.S. Pat. Nos. 5,970,973;5,672,581; 5,660,166; 5,404,871; and 5,450,336) or similar to currentlyused asthmatic devices, which deliver fixed and preset doses on eachactuation. Whichever type is used, it may be desirable to administer theinsulin in multiple actuations. By so doing, the patient can tailor hisintake according to the amount of carbohydrate he actually consumes,rather than the amount he expects to eat, by “topping up” his dose atsome time after beginning the meal. Furthermore, the more actuationsused to administer the insulin, the better the corresponding dosereliability (reproducibility), because inhalation administration tendsto vary from actuation to actuation, and multiple actuation delivery hasan averaging or smoothing effect.

To prevent hypoglycemia associated with using inhaled insulin fromoccurring between 2 and 5 hours after eating, a glucagon inhaler is usedto administer a s.c. dose equivalent of more than 5 to 16 ng/kg/min. ofglucagon administered through inhalation between hours 2 and 5 followingthe meal. In one embodiment, different inhalers for each type of insulinand for glucagon are used. In one embodiment, a unitary inhaler with atleast 2 drug chambers (for prandial insulin, glucagon and/or optionallybasal insulin) and capable of independent actuation is used.

B. Insulin Administered Parenterally

In accordance with Example 1.A.i, the patient administers his basal andprandial insulin parenterally. Because the risk of hypoglycemiaassociated with using LISPRO insulin typically occurs between 2 and 5hours after eating, the glucagon inhaler is used to administer a s.c.dose equivalent of 6 to 16 ng/kg/min. (i.e., an amount throughinhalation to get the same effect on blood glucose as though 6-16ng/kg/min. of glucagon administered subcutaneously) between hours 2 and5 following the meal. Alternatively, a modified glucagon of long-actingduration (e.g. iodinated glucagon) with delayed onset is used in theglucagon inhaler and administered at mealtimes with the prandialinsulin.

C. Insulin Administered by Pump

In accordance with Example 2.A, basal and prandial insulin are deliveredby pump. The risk of hypoglycemia arises after 2 to 3 hours, and so thepatient administers glucagon by inhaler 2 hours after eating. Headministers one puff from a metered dose inhaler at hours 2, 3 and 4,thus providing protection during the period of susceptibility. The doseper actuation corresponds to a s.c. dose equivalent amount of more than5 to 16 ng/kg/min. of glucagon. Alternatively, a modified glucagon oflong-acting duration (e.g. iodinated glucagon) with delayed onset isused in the glucagon inhaler and administered at mealtimes with theprandial insulin.

D. Insulin Administered Transdermally [Including Patch and TopicalCream]

In accordance with Example 3.A.ii, the patient administers his insulin(both basal and prandial) by transdermal patch or by topical cream. Therisk of hypoglycemia arises after 2 to 3 hours, and so the patientadministers glucagon by inhaler 2 hours after eating. He administers onepuff from a metered dose inhaler at hours 2, 3, and 4, thus providingprotection during the period of susceptibility. The dose per actuationcorresponds to a s.c. dose equivalent amount of more than 5 to 16ng/kg/min. glucagon. Alternatively, a modified glucagon of long-actingduration (e.g. iodinated glucagon) with delayed onset is used in theglucagon inhaler and administered at mealtimes with the prandialinsulin.

EXAMPLE 15 Co-Administration of Glucagon and Insulin, Admixed andParenterally for the Control of Diabetes and Prevention of Hypoglycemia

In Example 1, the insulin and glucagon were administered parenterallyand separately. In one embodiment, the two drugs are administeredsimultaneously in admixed form. Insulin and glucagon may be admixed withlittle if any interaction or degradation of either product. Innon-diabetics, it is typically found that following the increasedinsulin output after a meal of carbohydrate there is an associatedincrease in glucagon output (actually a restoration of output followingthe initial depression of glucagon output due to the initial gut-inducedrise in blood glucose after the ingestion of carbohydrate). This patternof insulin production followed by glucagon production assumes arelatively fixed relationship.

To ensure that the glucagon provides protection over the periodrequired, one can increase the amount of the glucagon component in theadmixture so that it is present in the required concentrations whendesired (to prevent hypoglycemia between 2 and 5 hours after the meal,in a s.c. dose equivalent of more than 5 to 30 ng/kg/min., andpreferably 8-16 ng/kg/min., or one can use a glucagon formulation withdelayed onset. In one embodiment, the formulation of glucagon has both adelayed release and an extended release (e.g., delayed by 2 to 3 hoursand releasing over approximately 3 hours). For example, any of theformulations discussed herein may be used.

In this example, an iodination method of increasing half life (asdescribed in U.S. Pat. No. 3,897,551; see form I3G) is employed. TheLISPRO insulin and I3Glucagon are admixed so that the modified glucagonis present at approximately 1.5% by weight of the insulin in the mixture(keeping the concentration of insulin per ml in our LISPRO formulationconstant). Because of the longer lasting effect of the modifiedglucagon, a smaller proportion of glucagon to insulin by weight will berequired.

The hypothetical patient then administers between 5 and 10 units(measured in terms of the insulin contained therein) of theinsulin-glucagon formulation at mealtimes in the standard way. In sodoing, he administers a s.c. dose equivalent of more than 5 to up to 16ng/kg/min. of modified glucagon. Given the longer action of the modifiedglucagon, this provides (assuming the modified glucagon has, forexample, twice the effect on glucose levels compared to standardglucagon) the same protection as described in Example 1.A. The glucagonso administered will be efficacious continuously between hours 2 and 5as required.

EXAMPLE 16 Co-Administration of Glucagon and Insulin, Admixed,Transdermally for the Control of Diabetes and Prevention of Hypoglycemia[Including Patch and Topical Cream]

In this example, both the insulin and glucagon are administered bytransdermal delivery. The prandial insulin and glucagon are admixed inthe same matrix or cream. Two different types of patch (or independentlyactuated compartments) can be employed. One patch (or compartment) willcontain a matrix designed to replace basal insulin over a 24 hourperiod. This patch can contain an amount of glucagon so that a s.c. doseequivalent of more than 5 to up to 20 ng/kg/min. of glucagon isdelivered to the patient. The other patch (or independently controlledcompartment) provides prandial glucagon and insulin in a single matrix.The onset times of the glucagon and insulin are matched so that when thepatch is actuated, insulin reaches efficacious plasma levels veryquickly whereas the glucagon only reaches efficacious levels after 2-3hours. The patch is applied at mealtimes and preferably no more than onehour before the meal.

In one embodiment, a unitary device containing four independentlyactuable compartments is used, one containing basal insulin, which isactivated on application and left active for 24 hours, and the other 3compartments containing the prandial insulin and glucagon in the samematrix, each compartment being separately activated at (or near)mealtimes and deactivated at some time after the meal, the time ofactivation being proportional to the amount of carbohydrate consumed. Onbeginning a meal (or up to an hour before the meal), the patientactivates one of these prandial compartments (e.g., by pulling away ahermetic plastic seal between the patch and the skin), a process whichinitiates the transdermal infusion of the admixed insulin and glucagon.At some later time, a period in direct proportion to the amount ofcarbohydrate taken, the prandial compartment is deactivated (e.g. byreplacing the barrier used to activate the compartment or the totalremoval of that compartment from the end of the patch).

The combined insulin and glucagon formulation in the prandialcompartment contains short acting insulin, and the patch is designed forrapid onset of the insulin. The glucagon component is designed to reachefficacious concentrations in the bloodstream between 1 and 3 hoursafter activation of the compartment, hence providing protection fromhypoglycemia at the appropriate part of the cycle.

In an alternative embodiment, a unitary device which allows for morethan 3 meals a day can be used and contains more than 3 prandialcompartments. The basal patch is designed to replace basal insulin (wornfor 24 hours before being replaced). The insulin used may be any insulinsuitable for transdermal delivery. It may be advantageous to useintermediate duration insulin in preference to short-acting insulin sothat any variation in insulin absorption over the lifetime of the patchwould be minimized by the relatively long lifetimes of the insulininvolved. The basal insulin compartment may also optionally contain anamount of glucagon (admixed) sufficient to supply basal glucagon overeach 24 hour period. This would have the beneficial effect of providingprotection from hypoglycemia throughout the day and in particular duringsleep.

EXAMPLE 17 Co-Administration of Glucagon and Insulin, Admixed, byInhalation for the Control of Diabetes and Prevention of Hypoglycemia[Including Pulmonary, Buccal, Nasal and Sublingual Delivery]

The present embodiment provides methods and pharmaceutical formulationsfor delivery of glucagon admixed with insulin by inhalation. In thisexample, a long acting glucagon (such as, for example, iodinatedglucagon as described in U.S. Pat. No. 3,897,551, e.g. I2G, or a zincprotamine glucagon) is admixed with LISPRO insulin and delivered by atypical insulin inhaler (e.g. as disclosed in patent U.S. Pat. No.5,970,973). Basal insulin may be delivered in the standard way bysubcutaneous injection, as described in Example 1A, or it may bedelivered by inhaler. Glucagon may optionally be included in thisformulation in an extended release formulation if desired to providebasal glucagon replacement.

The insulin powder used is admixed with the modified glucagon so thatthe modified glucagon content is a s.c. dose equivalent of more than 5to 20 ng/kg/min., and more preferably between 8 and 16 ng/kg/min. for1-3 units of insulin used. Proportionally larger amounts of glucagon canbe used when larger amounts of insulin are used (although the amount ofglucagon can stay constant regardless of the amount of insulin used).The amount can be adjusted as need, in light of the results from theprevious examples for this particular embodiment. The patient willadminister the combined insulin and glucagon at mealtimes to providesystemic insulin equivalent to between 5 and 10 units.

Although the present invention has been described in detail withreference to specific embodiments, those of skill in the art willrecognize that modifications and improvements are within the scope andspirit of the invention, as set forth in the claims that follow. Allpublications and patent documents cited herein are incorporated hereinby reference as if each such publication or document was specificallyand individually indicated to be incorporated herein by reference.Citation of publications and patent documents is not intended as anadmission that any such document is pertinent prior art, nor does itconstitute any admission as to the contents or date of the same.Definitions provided herein control over definitions found in the citedreferences or elsewhere. The invention having now been described by wayof written description and example, those of skill in the art willrecognize that the invention can be practiced in a variety ofembodiments and that the foregoing description and examples are forpurposes of illustration and not limitation of the following claims.

1. A pharmaceutical formulation comprising: insulin in an amounteffective for the control of diabetes; and glucagon in an amounteffective for the prevention of hypoglycemia in a human or other mammal,wherein said pharmaceutical formulation is configured to be administeredsubcutaneously, and wherein a ratio of insulin to glucagon is about 1unit of insulin to between more than 40 milliunits to 200 milliunits ofglucagon.
 2. The pharmaceutical composition of claim 1, wherein theamount of glucagon is between about 50 and 100 milliunits.
 3. Thepharmaceutical composition of claim 1, wherein the glucagon is alonger-acting form of glucagon.
 4. The pharmaceutical composition ofclaim 3, wherein the longer-acting form of glucagon contains iodine. 5.The pharmaceutical composition of claim 3, wherein the longer-actingform of glucagon contains zinc.
 6. The pharmaceutical composition ofclaim 5, wherein the longer-acting form of glucagon further comprisesprotamine.
 7. A method of treating diabetes in a human or other mammalwithout inducing hypoglycemia, said method comprising: administeringinsulin in an amount therapeutically effective for the control ofdiabetes, wherein said insulin is in an amount between 0.5 and 20 Unitsof insulin; and administering glucagon in time and an amounttherapeutically effective for the prevention of hypoglycemia, whereinsaid glucagon is administered subcutaneously, and wherein the amount ofglucagon administered is between more than 5 and less than or equal to100 ng per kg of patient per minute of desired glucagon effectiveness.8. The method of claim 7, wherein the amount of glucagon administered isbetween 6 and less than 18 ng per kg of patient per minute of desiredglucagon effectiveness.
 9. The method of claim 7, wherein said glucagonis a glucagon with a prolonged duration of action.
 10. The method ofclaim 7, wherein said glucagon is contained in a liposomal formulation.11. The method of claim 7, wherein said glucagon is contained in amicrosphere.
 12. The method of claim 7, comprising administering aformulation comprising both insulin and glucagon.
 13. The method ofclaim 7, wherein said insulin and glucagon are contained in a pump thatcontrols administration of a drug to a patient.
 14. The method of claim13, wherein said glucagon is administered simultaneously with insulin.15. The method of claim 14, wherein a ratio of glucagon to insulin isabout more than 40 to 200 milliunits of glucagon to 1 unit of insulin.16. The method of claim 15, wherein 2 units of insulin are administered.17. The method of claim 7, wherein 10 units of insulin are administeredand between 30 and 90 ng per kg of patient per minute of glucagon areadministered subcutaneously.
 18. A kit for the administration ofglucagon and insulin in amounts to prevent hypoglycemia, said kitcomprising: glucagon; insulin, wherein said glucagon and insulin are ina ratio of 1-20 units of insulin to 32-480 milliunits of glucagon; ameans for administering glucagon subcutaneously; and instructions forthe administration of insulin and glucagon so that the glucagon preventsa hypoglycemic event.
 19. The kit of claim 18, wherein the concentrationof glucagon when completely dissolved in the glycerine solution is morethan 500 micrograms per milliliter but less than 2000 micrograms permilliliter.
 20. The kit of claim 18, wherein said glucagon and insulinare in a ratio of 1-3 units of insulin to 32-96 milliunits of glucagon.21. The kit of claim 18, wherein the means for administering theglucagon subcutaneously is a pump and said pump is configured to deliverbetween about 6 to 20 ng/kg/minute of glucagon.